Adenoviral vectors for gene therapy containing deletions in the adenoviral genome

ABSTRACT

Adenoviral vectors which contain deletions of the early regions and/or late genes provide efficient delivery and expression of foreign nucleic acids of interest to patients. These vectors have a particular use in the treatment of cystic fibrosis patients. Furthermore, PAV vectors provide for a second generation of adenoviral vectors that contain the 5&#39; ITR&#39;s, the packaging signal and the E1A enhancer. Other adenoviral vectors contain a deletion of the E1 region or a deletion of E4 but retain orf3 or orf6, and can either retain or delete the E3 region.

RELATED APPLICATIONS

This is a continuation application(s) Ser. No. 08/136,742 filed on Oct.13, 1993, now U.S. Pat. No. 5,670,488, which is a continuation-in-partof application U.S. Ser. No. 07/985,478 filed Dec. 3, 1992, nowabandoned.

BACKGROUND OF THE INVENTION

Cystic Fibrosis (CF) is the most common fatal genetic disease in humans(Boat, T. F. et al. in The Metabolic Basis of Inherited Diseases(Scriver, C. R. et al. eds., McGraw-Hill, N.Y. (1989)). Approximatelyone in every 2,500 infants in the United States is born with thedisease. At the present time, there are approximately 30,000 CF patientsin the United States. Despite current standard therapy, the median ageof survival is only 26 years. Disease of the pulmonary airways is themajor cause of morbidity and is responsible for 95% of the mortality.The first manifestation of lung disease is often a cough, followed byprogressive dyspnea. Tenacious sputum becomes purulent because ofcolonization of Staphylococcus and then with Pseudomonas. Chronicbronchitis and bronchiectasis can be partially treated with currenttherapy, but the course is punctuated by increasingly frequentexacerbations of the pulmonary disease. As the disease progresses, thepatient's activity is progressively limited. End-stage lung disease isheralded by increasing hypoxemia, pulmonary hypertension, and corpulnonale.

The upper airways of the nose and sinuses are also involved by CF. Mostpatients with CF develop chronic sinusitis. Nasal polyps occur in 15-20%of patients and are common by the second decade of life.Gastrointestinal problems are also frequent in CF; infants may suffermeconium ileus. Exocrine pancreatic insufficiency, which producessymptoms of malabsorption, is present in the large majority of patientswith CF. Males are almost uniformly infertile and fertility is decreasedin females.

Based on both genetic and molecular analyses, a gene associated with CFwas isolated as part of 21 individual cDNA clones and its proteinproduct predicted (Kerem, B. S. et al. (1989) Science 245:1073-1080;Riordan, J. R. et al. (1989) Science 245:1066-1073; Rommens, J. M. etal. (1989) Science 245:1059-1065)). U.S. Ser. No. 07/488,307 describesthe construction of the gene into a continuous strand, expression of thegene as a functional protein and confirmation that mutations of the geneare responsible for CF. (See also Gregory, R. J. et al. (1990) Nature347:382-386; Rich, D. P. et al. (1990) Nature 347:358-362). Theco-pending patent application also discloses experiments which show thatproteins expressed from wild type but not a mutant version of the cDNAcomplemented the defect in the cAMP regulated chloride channel shownpreviously to be characteristic of CF.

The protein product of the CF associated gene is called the cysticfibrosis transmembrane conductance regulator (CFTR) (Riordan, J. R. etal. (1989) Science 245:1066-1073). CFTR is a protein of approximately1480 amino acids made up of two repeated elements, each comprising sixtransmembrane segments and a nucleotide binding domain. The two repeatsare separated by a large, polar, so-called R-domain containing multiplepotential phosphorylation sites. Based on its predicted domainstructure, CFTR is a member of a class of related proteins whichincludes the multi-drug resistance (MDR) or P-glycoprotein, bovineadenyl cyclase, the yeast STE6 protein as well as several bacterialamino acid transport proteins (Riordan, J. R. et al. (1989) Science245:1066-1073; Hyde, S. C. et al. (1990) Nature 346:362-365). Proteinsin this group, characteristically, are involved in pumping moleculesinto or out of cells.

CFTR has been postulated to regulate the outward flow of anions fromepithelial cells in response to phosphorylation by cyclic AMP-dependentprotein kinase or protein kinase C (Riordan, J. R. et al. (1989) Science245:1066-1073; Welsh, 1986; Frizzell, R. A. et al. (1986) Science233:558-560; Welsh, M. J. and Liedtke, C. M. (1986) Nature 322:467; Li,M. et al. (1988) Nature 331:358-360; Hwang, T-C. et al. (1989) Science244:1351-1353).

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of mutations (Cutting, G. R. et al. (1990) Nature 346:366-369;Dean, M. et al. (1990) Cell 61:863-870; and Kerem, B-S. et al. (1989)Science 245:1073-1080; Kerem, B-S. et al. (1990) Proc. Natl. Acad, Sci.USA 87:8447-8451). Population studies have indicated that the mostcommon CF mutation, a deletion of the 3 nucleotides that encodephenylalanine at position 508 of the CFTR amino acid sequence (AF508),is associated with approximately 70% of the cases of cystic fibrosis.This mutation results in the failure of an epithelial cell chloridechannel to respond to cAMP (Frizzell R. A. et al. (1986) Science233:558-560; Welsh, M. J. (1986) Science 232:1648-1650.; Li, M. et al.(1988) Nature 331:358-360; Quinton, P. M. (1989) Clin. Chem.35:726-730). In airway cells, this leads to an imbalance in ion andfluid transport. It is widely believed that this causes abnormal mucussecretion, and ultimately results in pulmonary infection and epithelialcell damage.

Studies on the biosynthesis (Cheng, S. H. et al. (1990) Cell 63:827-834;Gregory, R. J. et al. (1991) Mol. Cell Biol. 11:3886-3893) andlocalization (Denning, G. M. et al. (1992) J. Cell Biol. 118:551-559 )of CFTR AF508, as well as other CFTR mutants, indicate that many CFTRmutant proteins are not processed correctly and, as a result, are notdelivered to the plasma membrane (Gregory, R. J. et al. (1991) Mol. CellBiol. 11:3886-3893). These conclusions are consistent with earlierfunctional studies which failed to detect cAMP-stimulated C1⁻ channelsin cells expressing CFTR AF508 (Rich, D. P. et al. (1990) Nature347:358-363; Anderson, M. P. et al. (1991) Science 251:679-682).

To date, the primary objectives of treatment for CF have been to controlinfection, promote mucus clearance, and improve nutrition (Boat, T. F.et al. in The Metabolic Basis of Inherited Diseases (Scriver, C. R. etal. eds., McGraw-Hill, New York (1989)). Intensive antibiotic use and aprogram of postural drainage with chest percussion are the mainstays oftherapy. However, as the disease progresses, frequent hospitalizationsare required. Nutritional regimens include pancreatic enzymes andfat-soluble vitamins. Bronchodilators are used at times. Corticosteroidshave been used to reduce inflammation, but they may produce significantadverse effects and their benefits are not certain. In extreme cases,lung transplantation is sometimes attempted (Marshall, S. et al. (1990)Chest 98:1488).

Most efforts to develop new therapies for CF have focused on thepulmonary complications. Because CF mucus consists of a highconcentration of DNA, derived from lysed neutrophils, one approach hasbeen to develop recombinant human DNase (Shak, S. et al. (1990) Proc.Natl. Sci. Acad USA 87:9188). Preliminary reports suggest thataerosolized enzyme may be effective in reducing the viscosity of mucus.This could be helpful in clearing the airways of obstruction and perhapsin reducing infections. In an attempt to limit damage caused by anexcessof neutrophil derived elastase, protease inhibitors have been tested.For example, alpha-1-antitrypsin purified from human plasma has beenaerosolized to deliver enzyme activity to lungs of CF patients(McElvaney, N. et al. (1991) The Lancet 337:392). Another approach wouldbe the use of agents to inhibit the action of oxidants derived fromneutrophils. Although biochemical parameters have been successfullymeasured, the long term beneficial effects of these treatments have notbeen established.

Using a different rationale, other investigators have attempted to usepharmacological agents to reverse the abnormally decreased chloridesecretion and increased sodium absorption in CF airways. Defectiveelectrolyte transport by airway epithelial is thought to alter thecomposition of the respiratory secretions and mucus (Boat, T. F. et al.in The Metabolic Basis of Inherited Diseases (Scriver, C. R. et al.eds., McGraw-Hill, New York (1989); Quinton, P. M. (1990) FASEB J4:2709-2717). Hence, pharmacological treatments aimed at correcting theabnormalities in electrolyte transport could be beneficial. Trials arein progress with aerosolized versions of the drug amiloride; amilorideis a diuretic that inhibits sodium channels, thereby inhibiting sodiumabsorption Initial results indicate that the drug, is safe and suggest aslight change in the rate of disease progression, as measured by lungfunction tests (Knowles, M. et al. (1990) N. Eng. J Med. 322: 1189-1194;App, E.(1990) Am. Rev. Respir. Dis. 141:605. Nucleotides, such as ATP orUTP, stimulate purinergic receptors in the airway epithelium. As aresult, they open a class of chloride channel that is different fromCFTR chloride channels. In vitro studies indicate that ATP and UTP canstimulate chloride secretion (Knowles. M. et al. (1991) N. Eng. J. Med.325:533). Preliminary trails to test the ability of nucleotides tostimulate secretion in vivo, and thereby correct the electrolytetransport abnormalities are underway.

Despite progress in therapy, cystic fibrosis remains a lethal disease,and no current therapy treats the basic defect. However, two generalapproaches may prove feasible. These are: 1) protein replacement therapyto deliver the wild type protein to patients to augment their defectiveprotein, and; 2) gene replacement therapy to deliver wild type copies ofthe CF associated gene. Since the most life threatening manifestationsof CF involve pulmonary complications, epithelial cells of the upperairways are appropriate target cells for therapy.

The feasibility of gene therapy has been established by introducing awild type cDNA into epithelial cells from a CF patient and demonstratingcomplementation of the hallmark defect in chloride ion transport (Rich,D. P. et al. (1990) Nature 347:358-363 ). This initial work involvedcells in tissue culture, however, subsequent work has shown that todeliver the gene to the airways of whole animals, defective adenovirusesmay be useful (Rosenfeld, (1992) Cell 68:143-155. However, the safetyand effectiveness of using defective adenoviruses remain to bedemonstrated.

SUMMARY OF THE INVENTION

In general, the instant invention relates to vectors for transferringselected genetic material of interest (e.g., DNA or RNA) to cells invivo. In preferred embodiments, the vectors are adenovirus-based.Advantages of adenovirus-based vectors for gene therapy are that theyappear to be relatively safe and can be manipulated to encode thedesired gene product and at the same time are inactivated in terms oftheir ability to replicate in a normal lytic viral life cycle.Additionally, adenovirus has a natural tropism for airway epithelia.Therefore, adenovirus-based vectors are particularly preferred forrespiratory gene therapy applications such as gene therapy for cysticfibrosis.

In one embodiment, the adenovirus-based gene therapy vector comprises anadenovirus 2 serotype genome in which the E1a and E1b regions of thegenome, which are involved in early stages of viral replication havebeen deleted and replaced by genetic material of interest (e.g., DNAencoding the cystic fibrosis transmembrane regulator protein).

In another embodiment, the adenovirus-based therapy vector is apseudo-adenovirus (PAV). PAVs contain no potentially harmful viralgenes, have a theoretical capacity for foreign material of nearly 36 kb,may be produced in reasonably high titers and maintain the tropism ofthe parent adenovirus for dividing and non-dividing human target celltypes. PAVs comprise adenovirus inverted terminal repeats and theminimal sequences of a wild-type adenovirus type 2 genome necessary forefficient replication and packaging by a helper virus and geneticmaterial of interest. In a preferred embodiment, the PAV containsadenovirus 2 sequences.

In a further embodiment, the adenovirus-based gene therapy vectorcontains the open readin frame 6 (ORE6) of adenovira ear region 4 (E4)from the E4 promoter and is deleted for all other E4 open readingframes. Optionally, this vector can include deletions in the E1 and/orE3 regions. Alternatively, the adenovirus-based gene therapy vectorcontains the open reading frame 3 (ORF3) of adenoviral E4 from the E4promoter and is deleted for all other E4 open reading frames. Again,optionally, this vector can include deletions in the E1 and/or E3regions. The deletion of non-essential open reading frames of E4increases the cloning capacity by approximately 2 kb withoutsignificantly reducing the viability of the virus in cell culture. Incombination with deletions in the E1 and/or E3 regions of adenovirusvectors, the theoretical insert capacity of the resultant vectors isincreased to 8-9 kb.

The invention also relates to methods of gene therapy using thedisclosed vectors and genetically engineered cells produced by themethod.

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS

Further understanding of the invention may be had by reference to thetables and figures wherein:

FIG. 1 shows alignment of CFTR partial cDNA clones used in constructionof cDNA containing complete coding sequence of the CFTR; onlyrestriction sites relevant to the DNA constructions described below areshown.

FIG. 2 depicts plasmid construction of the CFTR cDNA clone pKK-CFTR1.

FIG. 3 depicts plasmid construction of the CFTR cDNA clone pKK-CFTR2.

FIG. 4 depicts plasmid construction of the CFTR cDNA clone pSC-CFTR2.

FIG. 5 shows a plasmid map of the CFTR cDNA clone pSC-CFTR2.

FIG. 6 shows the DNA sequence of synthetic DNAs used for insertion of anintron into the CFTR cDNA sequence, with the relevant restrictionendonuclease sites and nucleotide positions noted.

FIGS. 7A and 7B depict plasmid construction of the CFTR cDNA clonepKK-CFTR3.

FIG. 8 shows a plasmid map of the CFTR cDNA pKK-CFTR3 containing anintron between nucleotides 1716 and 1717.

FIG. 9 shows treatment of CFTR with glycosidases.

FIGS. 10A and 10B show an analysis of CFTR expressed from COS-7transfected cells.

FIGS. 11A and 11B show pulse-chase labeling of wild type and ΔF508mutant CFTR in COS-7 transfected cells.

FIGS. 12A-12D show immunolocalization of wild type and ΔF508 mutant CFTRin COS-7 cells transfected with pMT-CFTR or pMT-CFTR-ΔF508.

FIG. 13 shows an analysis of mutant forms of CFTR.

FIG. 14 shows a map of the first generation adenovirus based vectorencoding CFTR (Ad2/CFTR-1).

FIG. 15 shows the plasmid construction of the Ad2/CFTR- 1 vector.

FIGS. 16A and 16B show a map of the second generation adenovirus basedvector, PAV.

FIGS. 17A and 17B show the plasmid construction for a second generationadenoviral vector (Ad2E40RF6).

FIGS. 18A and 18B show differential cell analyses of bronchoalveolarlavage specimens from control and infected rats. These data demonstratethat none of the rats treated with Ad2/CFTR-1 had a change in the totalor differential white blood cell count 4, 10, and 14 days afterinfection (FIG. 18A) and 3, 7, 14 days after infection (FIG. 18B).

FIGS. 19A and 19B show examples of UV fluorescence from an agarose gelelectrophoresis, stained with ethidium bromide, of products of RT-PCRfrom nasal brushings of Rhesus monkeys after application of Ad2/CFTR-1or Ad2/β-Gal.

FIGS. 20A-20D show serum antibody titers in Rhesus monkeys after threevector administrations. These graphs demonstrate that all three monkeystreated with Ad2/CFTR-1 developed antibodies against adenovirus.

FIGS. 21A-21I are photomicrographs of human nasal mucosa immediatelybefore, during, and after Ad2/CFTR-1 application. These photomicrographsdemonstrate that inspection of the nasal mucosa showed mild to moderateerythema, edema, and exudate in patients treated with Ad2/CFTR-1 (FIGS.21A-21C) and in control patients (FIGS. 21G-21I). These changes wereprobably due to local anesthesia and vasoconstriction because when anadditional patient was exposed to Ad2/CFTR-1 in a method which did notrequire the use of local anesthesia or vasoconstriction, there were nosymptoms and the nasal mucosa appeared normal (FIGS. 21 D-21F).

FIG. 22 is a photomicrograph of a hematoxylin and eosin stained biopsyof human nasal mucosa obtained from the third patient three days afterAd2/CFTR-1 administration. This section shows a morphology consistentwith CF, i.e., a thickened basement membrane and occasionalmorphonuclear cells in the submucosa, but no abnormalities that could beattributed to the adenovirus vector.

FIG. 23 shows transepithelial voltage (Vt) across the nasal epitheliumof a normal human subject. Amiloride (μM) and terbutaline (μM) wereperfused onto the mucosal surface beginning at the times indicated.Under basal conditions Vt was electrically negative. Perfusion ofamiloride onto the mucosal surface inhibited Vt by blocking apical Na⁺channels.

FIGS. 24A and 24B show transepithelial voltage (Vt) across the nasalepithelium of normal human subjects (FIG. 24A) and patients with CF(FIG. 24B). Values were obtained under basal conditions, duringperfusion with amiloride (μM) and during perfusion of amiloride plusterbutaline (μM) onto the mucosal surface. Data are from seven normalsubjects and nine patients with CF. In patients with CF, Vt was moreelectrically negative than in normal subjects (FIG. 24B). Amilorideinhibited Vt in CF patients, as it did in normal subjects. However, Vtfailed to hyperpolarize when terbutaline was perfused onto theepithelium in the presence of amiloride. Instead, Vt either did notchange or became less negative, a result very different from thatobserved in normal subjects.

FIGS. 25A and 25B show transepithelial voltage (Vt) across the nasalepithelium of a third patient before (FIG. 25A) and after (FIG. 25B)administration of approximately 25 MOI of Ad2/CFTR-1. Amiloride andterbutaline were perfused onto the mucosal surface beginning at thetimes indicated. FIG. 25A shows an example from the third patient beforetreatment. FIG. 25B shows that in contrast to the response beforeAd2/CFTR-1 was applied, after virus replication, in the presence ofamiloride, terbutaline stimulated Vt.

FIGS. 26A-26F show the time of course changes in transepithelialelectrical properties before and after administration of Ad2/CFTR-1.FIGS. 26A and 26B are from the first patient who received approximately1 MOI; FIGS. 26C and 26D are from the second patient who receivedapproximately 3 MOI; and FIGS. 26E and 26F are from the third patientwho received approximately 25 MOI. FIGS. 26A, 26C, and 26E show valuesof basal transepithelial voltage (Vt) and FIGS. 26B, 26D, and 26F showthe change in transepithelial voltage (ΔVt) following perfusion ofterbutaline in the presence of amiloride. Day zero indicates the day ofAd2/CFTR-1 administration. FIGS. 26A, 26C, and 26E show the time courseof changes in basal Vt for all three patients. The decrease in basal Vtsuggests that application of Ad2/CFTR-1 corrected the CF electrolytetransport defect in nasal epithelium of all three patients. Additionalevidence came from an examination of the response to terbutaline. FIGS.26B, 26D, and 26F show the time course of the response. These dataindicate that Ad2/CFTR-1 corrected the CF defect in Cl⁻ transport.

FIGS. 27A and 27B show the time course of changes in transepithelialelectrical properties before and after administration of saline insteadof Ad2/CFTR-1 to CF patients. Day zero indicates the time of mockadministration. The top graph shows basal transepithelial voltage (Vt)and the bottom graph shows the change in transepithelial voltagefollowing perfusion with terbutaline in the presence of amiloride (ΔVt).Closed symbols are data from two patients that received localanesthetic/vasoconstriction and placement of the applicator for thirtyminutes. Open symbol is data from a patient that received localanesthetic/vasoconstriction, but not placement of the applicator.Symptomatic changes and physical findings were the same as thoseobserved in CF patients treated with a similar administration procedureand Ad2/CFTR-1.

FIG. 28 is a schematic of Ad2-ORF6/PGK-CFTR which differs fromAd2/CFTR-1 in that the latter utilized the endogenous E1a promoter, hadno poly A addition signal directly downstream of CFTR and retained anintact E4 region.

FIG. 29 shows short-circuit currents from CF nasal polyp epithelialcells infected with Ad2-ORF6/PGK-CFTR at multiplicities of 0.3, 3, and50. At the indicated times: (1) 10 μM amiloride, (2) cAMP agonists (10μM forskolin and 100 μM IBMX, and (3) 1 mM diphenylamine-2-carboxylatewere added to the mucosal solution.

FIGS. 30A-30C show summaries of the clinical signs (or lack thereof) ofinfection with Ad2-ORF6/PGK-CFTR.

FIGS. 31A-31C show a summary of blood counts, sedimentation rate, andclinical chemistries after infection with Ad2-ORF6/PGK-CFTR for monkeysC, D, and E. There was no evidence of a systemic inflammatory responseor other abnormalities of the clinical chemistries.

FIGS. 32A-32C show summaries of white blood cells counts in monkeys C,D, and E after infection with Ad2-ORF6/PGK-CFTR. These data indicatethat the administration of Ad2-ORF6/PGK-CFTR caused no change in thedistribution and number of inflammatory cells at any of the time pointsfollowing viral administration.

FIGS. 33A-33C show antibody titers to adenovirus prior to and after thefirst and second administrations of Ad2-ORF6/PGK-CFTR. Prior toadministration of Ad2-ORF6/PGK-CFTR, the monkeys had receivedinstillations of Ad2/CFTR- 1. Antibody titers measured by ELISA rosewithin one week after the first and second administrations ofAd2-ORF6/PGK-CFTR. Serum neutralizing antibodies also rose within a weekafter viral administration and peaked at day 24. No anti-adenoviralantibodies were detected by ELISA or neutralizing assay in nasalwashings of any of the monkeys.

DETAILED DESCRIPTION AND BEST MODE

Gene Therapy

As used herein, the phrase "gene therapy" refers to the transfer ofgenetic material (e.g., DNA or RNA) of interest into a host to treat orprevent a genetic or acquired disease or condition. The genetic materialof interest encodes a product (e.g., a protein polypeptide, peptide orfunctional RNA) whose production in vivo is desired. For example, thegenetic material of interest can encode a hormone, receptor, enzyme or(poly) peptide of therapeutic value. Examples of genetic material ofinterest include DNA encoding: the cystic fibrosis transmembraneregulator (CFTR), Factor VIII, low density lipoprotein receptor,beta-galactosidase, alpha-galactosidase, beta-glucocerebrosidase,insulin, parathyroid hormone, and alpha-1-antitrypsin.

Although the potential for gene therapy to treat genetic diseases hasbeen appreciated for many years, it is only recently that suchapproaches have become practical with the treatment of two patients withadenosine deamidase deficiency. The protocol consists of removinglymphocytes from the patients, stimulating them to grow in tissueculture, infecting them with an appropriately engineered retrovirusfollowed by reintroduction of the cells into the patient (Kantoff, P. etal. (1987) J Exp. Med 166:219). Initial results of treatment are veryencouraging. With the approval of a number of other human gene therapyprotocols for limited clinical use, and with the demonstration of thefeasibility of complementing the CF defect by gene transfer, genetherapy for CF appears a very viable option.

The concept of gene replacement therapy for cystic fibrosis is verysimple; a preparation of CFTR coding sequences in some suitable vectorin a viral or other carrier delivered directly to the airways of CFpatients. Since disease of the pulmonary airways is the major cause ofmorbidity and is responsible for 95% of mortality, airway epithelialcells are preferred target cells for CF gene therapy. The firstgeneration of CF gene therapy is likely to be transient and to requirerepeated delivery to the airways. Eventually, however, gene therapy mayoffer a cure for CF when the identity of the precursor or stem cell toair epithelial cells becomes known. If DNA were incorporated into airwaystem cells, all subsequent generations of such cells would makeauthentic CFTR from the integrated sequences and would correct thephysiological defect almost irrespective of the biochemical basis of theaction of CFTR.

Although simple in concept, scientific and clinical problems faceapproaches to gene therapy, not least of these being that CF requires anin vivo approach while all gene therapy treatments in humans to datehave involved ex vivo treatment of cells taken from the patient followedby reintroduction.

One major obstacle to be overcome before gene therapy becomes a viabletreatment approach for CF is the development of appropriate vectors toinfect tissue manifesting the disease and deliver the therapeutic CFTRgene. Since viruses have evolved very efficient means to introduce theirnucleic acid into cells, many approaches to gene therapy make use ofengineered defective viruses. However, the use of viruses in vivo raisessafety concerns. Although potentially safer, the use of simple DNAplasmid constructs containing mininal additional DNA, on the other hand,is often very inefficient and can result in transient proteinexpression.

The integration of introduced DNA into the host chromosome hasadvantages in that such DNA will be passed to daughter cells. In somecircumstances, integrated DNA may also lead to high or more sustainedexpression. However, integration often, perhaps always, requirescellular DNA replication in order to occur. This is certainly the casewith the present generation of retroviruses. This limits the use of suchviruses to circumstances where cell division occurs in a high proportionof cells. For cells cultured in vitro, this is seldom a problem,however, the cells of the airway are reported to divide onlyinfrequently (Kawanami, O. et al. (1979) An. Rev. Respir. Dis. 120:595).The use of retroviruses in CF will probably require damaging the airways(by agents such as SO₂ or O₃) to induce cell division. This may proveimpracticable in CF patients.

Even if efficient DNA integration could be achieved using viruses, thehuman genome contains elements involved in the regulation of cellulargrowth only a small fraction of which are presently identified. Byintegrating adjacent to an element such as a proto-oncogene or ananti-oncogene, activation or inactivation of that element could occurleading to uncontrolled growth of the altered cell. It is consideredlikely that several such activation/inactivation steps are usuallyrequired in any one cell to induce uncontrolled proliferation (R. A.Weinberg (1989) Cancer Research 49:3713 ), which may reduce somewhat thepotential risk. On the other hand, insertional mutagenesis leading totumor formation is certainly known in animals with some nondefectiveretroviruses (R. A. Weinberg (1989);Payne, G. S. et al. (1982) Nature295:209), and the large numbers of potential integrations occurringduring the lifetime of a patient treated repeatedly in vivo withretroviruses must raise concerns on the safety of such a procedure.

In addition to the potential problems associated with viral DNAintegration, a number of additional safety issues arise. Many patientsmay have preexisting antibodies to some of the viruses that arecandidates for vectors, for example, adenoviruses. In addition, repeateduse of such vectors might induce an immune response. The use ofdefective viral vectors may alleviate this problem somewhat, because thevectors will not lead to productive viral life cycles generatinginfected cells, cell lysis or large numbers of progeny viruses.

Other issues associated with the use of viruses are the possibility ofrecombination with related viruses naturally infecting the treatedpatient, complementation of the viral defects by simultaneous expressionof wild type virus proteins and containment of aerosols of theengineered viruses.

Gene therapy approaches to CF will face many of the same clinicalchallenges at protein therapy. These include the inaccessibility ofairway epithelium caused by mucus build-up and the hostile nature of theenvironment in CF airways which amy inactivate viruses/vectors. Elementsof the vector carriers may be immunogenic and introduction of the DNAmay be inefficient. These problems, as with protein therapy, areexacerbated by the absence of good animal model for the disease nor asimple clinical end point to measure the efficacy of treatment

CF Gene Therapy Vectors--Possible Options

Retroviruses--Although defective retroviruses are the best characterizedsystem and so far the only one approved for use in human gene therapy(Miller, A. D. (1990) Blood 76:271), the major issue in relation to CFis the requirement for dividing cells to achieve DNA integration andgene expression. Were conditions found to induce airway cell division,the in vivo application of retroviruses, especially if repeated overmany years, would necessitate assessment of the safety aspects ofinsertional mutagenesis in this context.

Adeno-Associated Virus--(AAV) is a naturally occurring defective virusthat requires other viruses such as adenoviruses or herpes viruses ashelper viruses(Muzyczka, N. (1992) in Current Topics in Microbiology andImmunology 158:97). It is also one of the few viruses that may integrateits DNA into non-dividing cells, although this is not yet certain.Vectors containing as little as 300 base pairs of AAV can be packagedand can integrate, but space for exogenous DNA is limited to about 4.5kb. CFTR DNA may be towards the upper limit of packaging. Furthermore,the packaging process itself is presently inefficient and safety issuessuch as immunogenecity, complementation and containment will also applyto AAV. Nevertheless, this system is sufficiently promising to warrantfurther study.

Plasmid DNA--Naked plasmid can be introduced into muscle cells byinjection into the tissue. Expression can extend over many months butthe number of positive cells is low (Wolff, J. et al. (1989) Science247:1465). Cationic lipids aid introduction of DNA into some cells inculture (Felgner, P. and Ringold, G. M. (1989) Nature 337:387).Injection of cationic lipid plasmid DNA complexes into the circulationof mice has been shown to result in expression of the DNA in lung(Brigham, K. et al. (1989) Am. J Med. Sci. 298:278). Instillation ofcationic lipid plasmid DNA into lung also leads to expression inepithelial cells but the efficiency of expression is relatively low andtransient (Hazinski, T. A. et al. (1991) Am. J Respir., Cell Mol. Biol.4:206). One advantage of the use of plasmid DNA is that it can beintroduced into non-replicating cells. However, the use of plasmid DNAin the CF airway environment, which already contains high concentrationsof endogenous DNA may be problematic.

Receptor Mediated Entry--In an effort to improve the efficiency ofplasmid DNA uptake, attempts have been made to utilize receptor-mediatedendocytosis as an entry mechanisms and to protect DNA in complexes withpolylysine (Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621). Onepotential problem with this approach is that the incoming plasmid DNAenters the pathway leading from endosome to lysosome, where muchincoming material is degraded. One solution to this problem is the useof transferrin DNA-polylysine complexes linked to adenovirus capsids(Curiel, D. T. et al. (1991) Proc. Natl. Acad Sci. USA 88:8850). Thelatter enter efficiently but have the added advantage of naturallydisrupting the endosome thereby avoiding shuttling to the lysosome. Thisapproach has promise but at present is relatively transient and suffersfrom the same potential problems of immunogenicity as other adenovirusbased methods.

Adenovirus--Defective adenoviruses at present appear to be a promisingapproach to CF gene therapy (Berkner, K. L. (1988) BioTechniques 6:616).Adenovirus can be manipulated such that it encodes and expresses thedesired gene product, (e.g., CFTR), and at the same time is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.In addition, adenovirus has a natural tropism for airway epithelial. Theviruses are able to infect quiescent cells as are found in the airways,offering a major advantage over retroviruses. Adenovirus expression isachieved without integration of the viral DNA into the host cellchromosome, thereby alleviating concerns about insertional mutagenesis.Furthermore, adenoviruses have been used as live enteric vaccines formany years with an excellent safety profile (Schwartz, A. R. et al.(1974) Am. Rev. Respir. Dis. 109:233-238). Finally, adenovirus mediatedgene transfer has been demonstrated in a number of instances includingtransfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats(Rosenfeld, M. A. et al. (1991) Science 252:431434; Rosenfeld et al.,(1992) Cell 68:143-155). Furthermore, extensive studies to attempt toestablish adenovirus as a causative agent in human cancer were uniformlynegative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

The following properties would be desirable in the design of anadenovirus vector to transfer the gene for CFTR to the airway cells of aCF patient. The vector should allow sufficient expression of the CFTR,while producing minimal viral gene expression. There should be minimalviral DNA replication and ideally no virus replication. Finally,recombination to produce new viral sequences and complementation toallow growth of the defective virus in the patient should be minimized.A first generation adenovirus vector encoding CFTR (Ad2/CFTR), made asdescribed in the following Example 7, achieves most of these goals andwas used in the human trials described in Example 10.

FIG. 14 shows a map of Ad2/CFR-1. As can be seen from the figure, thisfirst generation virus includes viral DNA derived from the commonrelatively benign adenovirus 2 serotype. The E1a and E1b regions of theviral genome, which are involved in early stages of viral replicationhave been deleted. Their removal impairs viral gene expression and viralreplication. The protein products of these genes also have immortalizingand transforming function in some non-permissive cells.

The CFTR coding sequence is inserted into the viral genome in place ofthe E1a/E1b region and transcription of the CFTR sequence is driven bythe endogenous E1a promoter. This is a moderately strong promoter thatis functional in a variety of cells. In contrast to some adenovirusvectors (Rosenfeld, M. et al. (1992) Cell 68:143), this adenovirusretains the E3 viral coding region. As a consequence of the inclusion ofE3, the length of the adenovirus-CFTR DNA is greater than that of thewild-type adenovirus. The greater length of the recombinant viral DNArenders it more difficult to package. This means that the growth of theAd2/CFTR virus is impaired even in permissive cells that provide themissing E1a and E1b functions.

The E3 region of the Ad2/CFTR-1 encodes a variety of proteins. One ofthese proteins, gp19, is believed to interact with and preventpresentation of class 1 proteins of the major histocompatability complex(MHC) (Gooding, C. R. and Wold, W. S. M. (1990) Crit. Rev. Immunol.10:53). This property prevents recognition of the infected cells andthus may allow viral latency. The presence of E3 sequences, therefore,has two useful attributes; first, the large size of the viral DNArenders it doubly defective for replication (i.e., it lacks earlyfunctions and is packaged poorly) and second, the absence of MHCpresentation could be useful in later applications of Ad2/CFTR-1 in genetherapy involving multiple administrations because it may avoid animmune response to recombinant virus containing cells.

Not only are there advantages associated with the presence of E3; theremay be disadvantages associated with its absence. Studies of E3 deletedvirus in animals have suggested that they result in a more severepathology (Gingsberg, H. S. et al. (1989) Proc. Natl. Acad. Sci. (USA)86:3823). Furthermore, E3 deleted virus, such as might be obtained byrecombination of an E1 plus E3 deleted virus with wild-type virus, isreported to outgrow wild-type in tissue culture (Barkner, K. L. andSharp, P. (1983) Nucleic Acids Research 11:6003). By contrast, however,a recent report of an E3 replacement vector encoding hepatitis B surfaceantigen, suggests that when delivered as a live enteric vaccine, such avirus replicates poorly in human compared to wild-type.

The adenovirus vector (Ad2/CFTR-1) and a related virus encoding themarker β-galactosidase (Ad2/β-gal) have been constructed and grown inhuman 293 cells. These cells contain the E1 region of adenovirus andconstitutively express E1a and E1b, which complement the defectiveadenoviruses by providing the products of the genes deleted from thevector. Because the size of its genome is greater than that of wild-typevirus, Ad2/CFTR is relatively difficult to produce.

The Ad2/CFTR-1 virus has been shown to encode CFTR by demonstrating thepresence of the protein in 293 cells. The Ad2/β-gal virus was shown toproduce its protein in a variety of cell lines grown in tissue cultureincluding a monkey bronchiolar cell line (4MBR-5), primary hamstertracheal epithelial cells, human HeLa, human CF PAC cells (see Example8) and airway epithelial cells from CF patients (Rich, O. et al. (1990)Nature 347:358).

Ad2/CFTR-1 is constructed from adenovirus 2 (Ad2) DNA sequences. Othervarieties of adenovirus (e.g., Ad3, Ad5, and Ad7) may also prove usefulas gene therapy vectors. This may prove essential if immune responseagainst a single serotype reduces the effectiveness of the therapy.

Second Generation Adenoviral Vectors

Adenoviral vectors currently in use retain most (≧80%) of the parentalviral genetic material leaving their safety untested and in doubtSecond-generation vector systems containing minimal adenoviralregulatory, packaging and replication sequences have therefore beendeveloped.

Pseudo-Adenovirus Vectors (PAV)-PAVs contain adenovirus invertedterminal repeats and the minimal adenovirus 5' sequences required forhelper virus dependent replication and packaging of the vector. Thesevectors contain no potentially harmful viral genes, have a theoreticalcapacity for foreign material of nearly 36 kb, may be produced inreasonably high titers and maintain the tropism of the parent virus fordividing and non-dividing human target cell types.

The PAV vector can be maintained as either a plasmid-borne construct oras an infectious viral particle. As a plasmid construct, PAV is composedof the minimal sequences from wild type adenovirus type 2 necessary forefficient replication and packaging of these sequences and any desiredadditional exogenous genetic material, by either a wild-type ordefective helper virus.

Specifically, PAV contains adenovirus 2 (Ad2) sequences as shown in FIG.17, from nucleotide (nt) 0-356 forming the 5' end of the vector and thelast 109 nt of Ad2 forming the 3' end of the construct. The sequencesincludes the Ad2 flanking inverted terminal repeats (5'ITR) and the 5'ITR adjoining sequences containing the known packaging signal and E1aenhancer. Various convenient restriction sites have been incorporatedinto the fragments, allowing the insertion of promoter/gene cassetteswhich can be packaged in the PAV virion and used for gene transfer (e.g.for gene therapy). The construction and propagation of PAV is describedin detail in the following Example 11. By not containing most nativeadenoviral DNA, the PAVs described herein are less likely to produce apatient immune reponse or to replicate in a host.

In addition, the PAV vectors can accomodate foreign DNA up to a maximumlength of nearly 36 kb. The PAV vectors therefore, are especially usefulfor cloning larger genes (e.g., CFTR (7.5 kb)); Factor VIII (8 kb);Factor IX (9 kb)), which, traditional vectors have difficultyaccomodating. In addition, PAV vectors can be used to transfer more thanone gene, or more than one copy of a particular gene. For example, forgene therapy of cystic fibrosis, PAVs can be used to deliver CFTR inconjunction with other genes such as anti proteases (e.g., antiproteasealpha-l-antitrypsin) tissue inhibitor of metaloproteinase, antioxidants(e.g., superoxide dismutase), enhancers of local host defense (e.g.,interferons), mucolytics (e.g., DNase); and proteins which blockinflammatory cytokines.

Ad2-E4/ORF6 Adenovirus Vectors

An adenoviral construct expressing only the open reading frame 6 (ORF6)of adenoviral early region 4 (E4) from the E4 promoter and which isdeleted for all other known E4 open reading frames was constructed asdescribed in detail in Example 12. Expression of E4 open reading frame 3is also sufficient to provide E4 functions required for DNA replicationand late protein synthesis. However, it provides these functions withreduced efficiency compared to expression of ORF6, which will likelyresult in lower levels of virus production. Therefore expressing ORF6,rather than ORF3, appears to be a better choice for producingrecombinant adenovirus vectors.

The E4 region of adenovirus is suspected to have a role in viral DNAreplication, late mRNA synthesis and host protein synthesis shut off, aswell as in viral assembly (Falgout, B. and G. Ketner (1987) J. Virol.61:3759-3768). Adenovirus early region 4 is required for efficient virusparticle assembly. Adenovirus early region 4 encodes functions requiredfor efficient DNA replication, late gene expression, and host cellshutoff. Halbert, D. N. et al. (1985) J. Virol. 56:250-257.

The deletion of non-essential open reading frames of E4 increases thecloning capacity of recombinant adenovirus vectors by approximately 2 kbof insert DNA without significantly reducing the viability of the virusin cell culture. When placed in combination with deletions in the E1and/or E3 regions of adenovirus vectors, the theoretical insert capacityof the resultant vectors is increased to 8-9 kb. An example of wherethis increased cloning capacity may prove useful is in the developmentof a gene therapy vector encoding CFTR. As described above, the firstgeneration adenoviral vector approaches the maximum packaging capacityfor viral DNA encapsidation. As a result, this virus grows poorly andmay occassionaly give rise to defective progeny. Including an E4deletion in the adenovirus vector should alleviate these problems. Inaddition, it allows flexibility in the choice of promoters to drive CFTRexpression from the virus. For example, strong promoters such as theadenovirus major late promoter, the cytomegalovirus immediate earlypromoter or a cellular promoter such as the CFTR promoter, which may betoo large for first-generation adenovirus can be used to driveexpression.

In addition, by expressing only ORF6 of E4, these second generationadenoviral vectors may be safer for use in gene therapy. Although ORF6expression is sufficient for viral DNA replication and late proteinsynthesis in immortalized cells, it has been suggested that ORF6/7 of E4may also be required in non-dividing primary cells (Hemstrom, C. et al.(1991) J. Virol. 65:1440-1449). The 19 kD protein produced from openreading frame 6 and 7 (ORF6/7) complexes with and activates cellulartranscription factor E2F, which is required for maximal activation ofearly region 2. Early region 2 encodes proteins required for viral DNAreplication. Activated transcription factor E2F is present inproliferating cells and is involved in the expression of genes requiredfor cell proliferation (e.g., DHFR, c-myc), whereas activated E2F ispresent in lower levels in non-proliferating cells. Therefore, theexpression of only ORF6 of E4 should allow the virus to replicatenormally in tissue culture cells (e.g., 293 cells), but the absence ofORF6/7 would prevent the potential activation of transcription factorE2F in non-dividing primary cells and thereby reduce the potential forviral DNA replication.

Target Tissue

Because 95% of CF patients die of lung disease, the lung is a preferredtarget for gene therapy. The hallmark abnormality of the disease isdefective electrolyte transport by the epithelial cells that line theairways. Numerous investigators (reviewed in Quinton, F. (1990) FASEB J.4:2709) have observed: a) a complete loss of cAMP-mediatedtransepithelial chloride secretion, and b) a two to three fold increasein the rate of Na+ absorption. cAMP-stimulated chloride secretionrequires a chloride channel in the apical membrane (Welsh, M. J. (1987)Physiol Rev. 67:1143-1184). The discovery that CFTR is aphosphorylation-regulated chloride channel and that the properties ofthe CFTR chloride channel are the same as those of the chloride channelsin the apical membrane, indicate that CFTR itself mediatestransepithelial chloride secretion. This conclusion was supported bystudies localizing CFTR in lung tissue: CFTR is located in the apicalmembrane of airway epithelial cells (Denning, G. M. et al. (1992) J.Cell BioL 118:551) and has been reported to be present in the submucosalglands (Taussig et al., (1973) J. Clin. Invest 89:339). As a consequenceof loss of CFTR function, there is a loss of cAMP-regulatedtransepithelial chloride secretion. At this time it is uncertain howdysfunction of CFTR produces an increase in the rate of Na+ absorption.However, it is thought that the defective chloride secretion andincreased Na+ absorption lead to an alteration of the respiratory tractfluid and hence, to defective mucociliary clearance, a normal pulmonarydefense mechanism. As a result, clearance of inhaled material from thelung is impaired and repeated infections ensue. Although the presumedabnormalities in respiratory tract fluid and mucociliary clearanceprovide a plausible explanation for the disease, a precise understandingof the pathogenesis is still lacking.

Correction of the genetic defect in the airway epithelial cells islikely to reverse the CF pulmonary phenotype. The identity of thespecific cells in the airway epithelium that express CFTR cannot beaccurately determined by immunocytochemical means, because of the lowabundance of protein. However, functional studies suggest that theciliated epithelial cells and perhaps nonciliated cells of the surfaceepithelium are among the main cell types involved in electrolytetransport. Thus, in practical terms, the present preferred target cellfor gene therapy would appear to be the mature cells that line thepulmonary airways. These are not rapidly dividing cells; rather, most ofthem are nonproliferating and many may be terminally differentiated. Theidentification of the progenitor cells in the airway is uncertain.

Although CFTR may also be present in submucosal glands (Trezise, A. E.and Buchwald, M. (1991) Nature 353:434; Englehardt, J. F. et al. (1992)J. Clin. Invest. 90:2598-2607), there is no data as to its function atthat site; furthermore, such glands appear to be relativelyinaccessible.

The airway epithelium provides two main advantages for gene therapy.First, access to the airway epithelium can be relatively noninvasive.This is a significant advantage in the development of deliverystrategies and it will allow investigators to monitor the therapeuticresponse. Second, the epithelium forms a barrier between the airwaylumen and the interstitium. Thus, application of the vector to the lumenwill allow access to the target cell yet, at least to some extent, limitmovement through the epithelial barrier to the interstitium and fromthere to the rest of the body.

Efficiency of Gene Delivery Required to Correct The Genetic Defect

It is unlikely that any gene therapy protocol will correct 100% of thecells that normally express CFTR. However, several observations suggestthat correction of a small percent of the involved cells or expressionof a fraction of the normal amount of CFTR may be of therapeuticbenefit.

a. CF is an autosomal recessive disease and heterozygotes have no lungdisease.

Thus, 50% of wild-type CFTR would appear sufficient for normal function.

b. This issue was tested in mixing experiments using CF cells andrecombinant CF cells expressing wild-type CFTR (Johnson, L. G. et al.(1992) Nature Gen 2:21). The data obtained showed that when anepithelium is reconstituted with as few as 6-10% of corrected cells,chloride secretion is comparable to that observed with an epitheliumcontaining 100% corrected cells. Although CFTR expression in therecombinant cells is probably higher than in normal cells, this resultsuggests that in vivo correction of all CF airway cells may not berequired.

c. Recent observations show that CFTR containing some CF-associatedmutations retains residual chloride channel activity (Sheppard, D. N. etal. (1992) Pediatr. Pulmon Suppl. 8:250; Strong, T. V. et al. (1991) N.Eng. J. Med. 325:1630). These mutations are associated with mild lungdisease. Thus, even a very low level of CFTR activity may at leastpartly ameliorate the electrolyte transport abnormalities.

d. As indicated in experiments described below in Example 8,complementation of CF epithelia, under conditions that probably wouldnot cause expression of CFTR in every cell, restored cAMP stimulatedchloride secretion.

e. Levels of CFTR in normal human airway epithelia are very low and arebarely detectable. It has not been detected using routine biochemicaltechniques such as immunoprecipitation or immunoblotting and has beenexceedingly difficult to detect with immunocytochemical techniques(Denning, G. M. et al. (1992) J. Cell Biol. 118:551). Although CFTR hasbeen detected in some cases using laser-scanning confocal microscopy,the signal is at the limits of detection and cannot be detected abovebackground in every case. Despite that minimal levels of CFTR, thissmall amount is sufficient to generate substantial cAMP-stimulatedchloride secretion. The reason that a very small number of CFTR chloridechannels can support a large chloride secretory rate is that a largenumber of ions can pass through a single channel (10⁶ -10⁷ ions/sec)(Hille, B. (1984) Sinauer Assoc. Inc., Sunderland, Mass. 420426).

f. Previous studies using quantitative PCR have reported that the airwayepithelial cells contain at most one to two transcripts per cell(Trapnell, B. C. et al. (1991) Proc. Natl. Acad Sci. USA 88:6565).

Gene therapy for CF would appear to have a wide therapeutic index. Justas partial expression may be of therapeutic value, overexpression ofwild-type CFTR appears unlikely to cause significant problems. Thisconclusion is based on both theoretical considerations and experimentalresults. Because CFTR is a regulated channel, and because it has aspecific function in epithelia it is unlikely that overexpression ofCFTR will lead to uncontrolled chloride secretion. First, secretionwould require activation of CFTR by cAMP-dependent phosphorylation.Activation of this kinase is a highly regulated process. Second, even ifCFTR chloride channels open in the apical membrane, secretion will notensue without regulation of the basolateral membrane transporters thatare required for chloride to enter the cell from the interstitial space.At the basolateral membrane, the sodium-potassium-chloride cotransporterand potassium channels serve as important regulators of transeptihelialsecretion (Welsh, M. J. (1987) Physio. Rev. 67:1143-1184).

Human CFTR has been expressed in transgenic mice under the control ofthe surfactant protein C(SPC) gene promoter (Whitesett, J. A. et al.(1992) Nature Gen. 2:13) and the casein promoter (Ditullio, P. et al(1992) Bio/Technology 10:74 ). In those mice, CFTR was overexpressed inbronchiolar and alveolar epithelial cells and in the mammary glands,respectively. Yet despite the massive overexpression in the transgenicanimals, there were no observable morphologic or functionalabnormalities. In addition, expression of CFTR in the lungs of cottonrats produced no reported abnormalities (Rosenfeld, M. A. et al. (1992)Cell 68:143-155).

The present invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting. Thecontents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1

Generation of Full Length CFTR cDNAs

Nearly all of the commonly used DNA cloning vectors are based onplasmids containing modified pMBI replication origins and are present atup to 500 to 700 copies per cell (Sambrook et al. Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press 1989). Thepartial CFTR cDNA clones isolated by Riordan et al. were maintained insuch a plasmid. It was postulated that an alternative theory tointrinsic clone instability to explain the apparent inability to recoverclones encoding full length CFTR protein using high cot number plasmids,was that it was not possible to clone large segments of the CFTR cDNA athigh gene dosage in E coli. Expression of the CFTR or portions of theCFTR from regulatory sequences capable of directing transcription and/ortranslation in the bacterial host cell might result in inviability ofthe host cell due to toxicity of the transcript or of the full lengthCFTR protein or fragments thereof. This inadvertent gene expressioncould occur from either plasmid regulatory sequences or crypticregulatory sequences within the recombinant CFTR plasmid which arecapable of functioning in E. coli. Toxic expression of the CFTR codingsequences would be greatly compounded if a large number of copies of theCFTR cDNA were present in cells because a high copy number plasmid wasused. If the product was indeed toxic as postulated, the growth of cellscontaining fall length and correct sequence would be activelydisfavored. Based upon this novel hypothesis, the following procedureswere undertaken. With reference to FIG. 2, partial CFTR clone T164.5 wascleaved with restriction enzymes Sph 1 and Pst 1 and the resulting 3.9kb restriction fragment containing exons 11 through most of exon 24(including an uncharacterized 119 bp insertion reported by Riordan etal. between nucleotides 1716 and 1717), was isolated by agarose gelpurification and ligated between the Sph 1 and Pst 1 sites of the pMB1based vector pkk223-3 (Brosius and Holy, (1984) Proc. Natl. Acad Sci81:6929). It was hoped that the pMB1 origin contained within thisplasmid would allow it and plasmids constructed from it to replicate at15-20 copies per host E. coli cell (Sambrook et al. Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press 1989). Theresultant plasmid clone was called pkk-4.5.

Partial CFTR clone T11 was cleaved with Eco R1 and Hinc II and the 1.9kb band encoding the first 1786 nucleotides of the CFTR cDNA plus anadditional 100 bp of DNA at the 5' end was isolated by agarose gelpurification. This restriction fragment was inserted between the Eco R1site and Sma 1 restriction site of the plamid Bluescript Sk-(Stratagene, catalogue number 212206), such that the CFTR sequences werenow flanked on the upstream (5') side by a Sal 1 site from the cloningvector. This clone, designated T11-R, was cleaved with Sal 1 and Sph 1and the resultant 1.8 kb band isolated by agarose gel purification.Plasmid pkk4.5 was cleaved with Sal 1 and Sph 1 and the large fragmentwas isolated by agarose gel purification. The purified T11-R fragmentand pkk4.5 fragments were ligated to construct pkk-CFTR1. pkk-CFTR1contains exons 1 through 24 of the CFTR cDNA. It was discovered thatthis plasmid is stably maintained in E. coli cells and confers nomeasureably disadvantageous growth characteristics upon host cells.

pkk-CFTR1 contains, between nucleotides 1716 and 1717, the 119 bp insertDNA derived from partial cDNA clone T164.5 described above. In addition,subsequent sequence analysis of pkk-CFTR1 revealed unreporteddifferences in the coding sequence between that portion of CFTR1 derivedfrom partial cDNA clone T11 and the published CFTR cDNA sequence. Theseundesired differences included a 1 base-pair deletion at position 995and a C to T transition at position 1507.

To complete construction of an intact correct CFTR coding sequencewithout mutations or insertions and with reference to the constructionscheme shown in FIG. 3, pkk-CFTR1 was cleaved with Xba I and Hpa I, anddephosphorylated with calf intestinal alkaline phosphatase. In addition,to reduce the likelihood of recovering the original clone, the smallunwanted Xba I/Hpa I restriction fragment from pKK-CFTR1 was digestedwith Sph I. T 16-1 was cleaved with Xba I and Acc I and the 1.15 kbfragment isolated by agarose gel purification. T164.5 was cleaved withAcc I and Hpa I and the 0.65 kb band was also isolated by agarose gelpurification. The two agarose gel purified restriction fragments and thedephosphorylated pKK-CFTR1 were ligated to produce pKK-CFTR2.Alternatively, pKK-CFTR2 could have been constructed using correspondingrestriction fragments from the partial CFTR cDNA clone C1-1/5. pKK-CFTR2contains the uninterrupted CFTR protein coding sequence and conferredslow growth upon E coli host cells in which it was inserted, whereaspKK-CFTR1 did not. The origin of replication of pKK-CFTR2 is derivedfrom pMB1 and confers a plasmid copy number of 15-20 copies per hostcell.

Example 2

Improving Host Cell Viability

An additional enhancement of host cell viability was accomplished by afurther reduction in the copy number of CFTR cDNA per host cell. Thiswas achieved by transferring the CFTR cDNA into the plasmid vector,pSC-3Z. pSC-3Z was constructed using the pSC101 replication origin ofthe low copy number plasmid pLG338 (Stoker et al., Gene 18, 335 (1982))and the ampicillin resistance gene and polylinker of pGEM-3Z (availablefrom Promega). pLG338 was cleaved with Sph I and pVu II and the 2.8 kbfragment containing the replication origin isolated by agarose gelpurification. pGEM-3Z was cleaved with ALw NI, the resultant restrictionfragment ends treated with T4 DNA polymerase and deoxynucleotidetriphosphates, cleaved with Sph I and the 1.9 kb band containing theampicillin resistance gene and the polylinker was isolated by agarosegel purification. The pLG338 and pGEM-3Z fragments were ligated togetherto produce the low copy number cloning vector pSC-3Z. pSC-3Z and otherplasmids containing pSC101 origins of replication are maintained atapproximately five copies per cell (Sambrook et al.).

With additional reference to FIG. 4, pKK-CFTR2 was cleaved with Eco RV,Pst I and Sal I and then passed over a Sephacryl S400 spun column(available from Pharmacia) according to the manufacturer's procedure inorder to remove the Sal I to Eco RV restriction fragment which wasretained within the column. pSC-3Z was digested with Sma I and Pst I andalso passed over a Sephacryl S400 spun column to remove the small SmaI/Pst I restriction fragment which was retained within the column. Thecolumn eluted fractions from the pKK-CFTR2 digest and the pSC-3Z digestwere mixed and ligated to produce pSC-CFTR2. A map of this plasmid ispresented in FIG. 5. Host cells containing CFTR cDNAs at this andsimilar gene dosages grow well and have stably maintained therecombinant plasmid with the full length CFTR coding sequence. Inaddition, this plasmid contains a bacteriophage T7 RNA polymerasepromoter adjacent to the CFTR coding sequence and is thereforeconvenient for in vitro transcription/translation of the CFTR protein.The nucleotide sequence of CFTR coding region from pSC-CFTR2 plasmid ispresented in Sequence Listing 1 as SEQ ID NO:1. Significantly, thissequence differs from the previously published (Riordan, J. R. et al.(1989) Science 245:1066-1073) CFTR sequence at position 1990, wherethere is C in place of the reported A. See Gregory, R. J. et al. (1990)Nature 347:382-386. E. coli host cells containing pSC-CFTR2, internallyidentified with the number pSC-CFTR2/AG1, have been deposited at theAmerican Type Culture Collection and given the accession number: ATCC68244.

Example 3

Alternate Method for Improving Host Cell Viability

A second method for enhancing host cell viability comprises disruptionof the CFTR protein coding sequence. For this purpose, a syntheticintron was designed for insertion between nucleotides 1716 and 1717 ofthe CFTR cDNA. This intron is especially advantageous because of itseasily manageable size. Furthermore, it is designed to be efficientlyspliced from CFTR primary RNA transcripts when expressed in eukaryoticcells. Four synthetic oligonucleotides were synthesized (1195RG, 1196RG,1197RG and 1198RG) collectively extending from the Sph I cleavage siteat position 1700 to the Hinc II cleavage site at position 1785 andincluding the additional 83 nucleotides between 1716 and 1717 (see FIG.6) SEQ ID NO. 10. These oligonucleotides were phosphorylated with T4polynucleotide kinase as described by Sambrook et al., mixed together,heated to 95° C. for 5 minutes in the same buffer used duringphosphorylation, and allowed to cool to room temperature over severalhours to allow annealing of the single stranded oligonucleotides. Toinsert the synthetic intron into the CFTR coding sequence and withreference to FIGS. 7A and 7B, a subclone of plasmid T11 was made bycleaving the B11 site in the polylinker, repairing the recessed ends ofthe cleaved DNA with deoxynucleotide triphosphates and the largefragment of DNA Polymerase I and religating the DNA. This plasmid wasthen digested with Eco RV and Nru I and religated. The resulting plasmidT6-A5' extended from the Nru I site at position 490 of the CFTR cDNA tothe 3' end of clone T16 and contained single sites for Sph I and Hinc IIat positions corresponding to nucleotides 1700 and 1785 of the CFTRcDNA. T16-Δ5' plasmid was cleaved with Sph I and Hinc II and the largefragment was isolated by agarose gel purification. The annealedsynthetic oligonucleotides were ligated into this vector fragment togenerate T16-intron.

T16-intron was then digested with Eco RI and Sma I and the largefragment was isolated by agarose gel purification. T164.5 was digestedwith Eco RI and Sca I and the 790 bp fragment was also isolated byagarose gel purification. The purified T16-intron and T16-4.5 fragmentswere ligated to produce T16-intron-2. T16-intron-2 contains CFTR cDNAsequences extending from the Nru I site at position 490 to the Sca Isite at position 2818, and includes the unique Hpa I site at position2463 which is not present in T16-1 or T16-intron-1.

T-16-intron-2 was then cleaved with Xba I and Hpa I and the 1800 bpfragment was isolated by agarose gel purification. pKK-CFTR1 wasdigested with Xba I and Hpa I and the large fragment was also isolatedby agarose gel purification and ligated with the fragment derived fromT16-intron-2 to yield pKK-CFTR3, shown in FIG. 8. The CFTR cDNA withinpKK-CFTR3 is identical to that within pSC-CFTR2 and pKK-CFTR2 except forthe insertion of the 83 bp intron between nucleotides 1716 and 1717. Theinsertion of this intron resulted in improved growth characteristics forcells harboring pKK-CFTR3 relative to cells containing the unmodifiedCFTR cDNA in pKK-CFTR2.

Example 4

In vitro Transcription/Translation

In addition to sequence analysis, the integrity of the CFTR cDNA openreading frame was verified by in vitro transcription/translation. Thismethod also provided the initial CFTR protein for identificationpurposes. 5 micrograms of pSC-CFTR2 plasmid DNA were linearized with SalI and used to direct the synthesis of CFTR RNA transcripts with T7 RNApolymerase as described by the supplier (Stratagene). This transcriptwas extracted with phenol and chloroform and precipitated with ethanol.The transcript was resuspended in 25 microliters of water and varyingamounts were added to a reticulocyte lysate in vitro translation system(Promega). The reactions were performed as described by the supplier inthe presence of canine pancreatic microsomal membranes (Promega), using³⁵ S-methionine to label newly synthesized proteins. In vitrotranslation products were analysed by discontinuous polyacrylamide gelelectrophoresis in the presence of 0.1% SDS with 8% separating gels(Laemmii, U. K. (1970) Nature 227:680-685). Before electrophoresis, thein vitro translation reactions were denatured with 3% SDS, 8M urea and5% 2-mercaptoethanol in 0.65M Tris-HCl, pH 6.8. Followingelectrophoresis, the gels were fixed in methanol:acetic acid:water(30:10:60), rinsed with water and impregnated with 1M sodium salicylate.³⁵ S labelled proteins were detected by fluorgraphy. A band ofapproximately 180 kD was detected, consistent with translation of thefull length CFTR insert.

Example 5

Elimination of Cryptic Regulatory Signals

Analysis of the DNA sequence of the CFTR has revealed the presence of apotential E. coli RNA polymerase promoter between nucleotides 748 and778 which conforms well to the derived consensus sequence for E. colipromoters (Reznikoff and McClure, Maximizing Gene Expression, 1,Butterworth Publishers, Stoneham, Mass.). If this sequence functions asa promoter functions in E. coli, it could direct synthesis ofpotentially toxic partial CFTR polypeptides. Thus, an additionaladvantageous procedure for maintaining plasmids containing CFTR cDNAs inE.coli would be to alter the sequence of this potential promoter suchthat it will not function in E. coli. This may be accomplished withoutaltering the amino acid sequence encoded by the CFTR cDNA. Specifically,plasmids containing complete or partial CFTR cDNA's would be altered bysite-directed mutagenesis using synthetic olignucleotides (Zoller andSmith, (1983) Methods Enzymol. 10:468 ). More specifically, altering thenucleotide sequence at position 908 from a T to C and at position 774from an A to a G effectively eliminates the activity of this promotersequence without altering the amino acid coding potential of the CFTRopen reading frame. Other potential regulatory signals within the CFTRcDNA for transcription and translation could also be advantageouslyaltered and/or deleted by the same method.

Futher analysis has identified a sequence extending from nucleotide 908to 936 which functions efficiently as a transcriptional promoter elementin E. coli (Gregory, R. J. et al. (1990) Nature 347:382-386). Mutationat position 936 is capable of inactivating this promoter and allowingthe CFTR cDNA to be stably maintained as a plasmid in E. coli (Cheng, S.H. et al. (1990) Cell 63:827-834). Specifically position 936 has beenaltered from a T to a C residue without the amino acid sequence encodedby the cDNA being altered Other mutations within this regulatory elementdescribed in Gregory, R. J. et al. (1990) Nature 347:382-386 could alsobe used to inactivate the transcriptional promoter activity.Specifically, the sequence from 908 to 913 (TTGTGA) and from 931 to 936(GAAAAT) could be altered by site directed mutagenesis without alteringthe amino acid sequence encoded by the cDNA.

Example 6

Cloning of CFTR in Alternate Host Systems

Although the CFTR cDNA displays apparent toxicity in E. coli cells,other types of host cells may not be affected in this way. Alternativehost systems in which the entire CFTR cDNA protein encoding region maybe maintained and/or expressed include other bacterial species andyeast. It is not possible a priori to predict which cells might beresistant and which might not. Screening a number of differenthost/vector combinations is necessary to find a suitable host tolerantof expression of the full length protein or potentially toxic fragmentsthereof.

Example 7

Generation of Adenovirus Vector Encoding CFTR (Ad2/CFTR)

1. DNA preparation--Construction of the recombinant Ad2/CFTR-1 virus(shown as SEQ ID NO:3) was accomplished as follows: The CFTR cDNA wasexcised from the plasmid pCMV-CFTR-936C using restriction enzymes 1 andEc11361. pCMV-CFTR-936C consists of a minimal CFTR cDNA encompassingnucleotides 123-4622 of the published CFTR sequence cloned into themultiple cloning site of pRC/CMV (Invitrogen Corp.) using syntheticlinkers. The CFTR cDNA within this plasmid has been completelysequenced. The Spel/EcII361 restriction fragment contains 47 bp of 5'sequence derived from synthetic linkers and the multiple cloning site ofthe vector.

The CFTR cDNA (the sequence of which is shown as SEQ ID NO:1 and theamino acid sequence encoded by the CFTR cDNA is shown as SEQ ID NO:2)was inserted between the NheI and SnaB1 restriction sites of theadenovirus gene transfer vector pBR-Ad2-7. pBR-Ad2-7 is a pBR322 basedplasmid containing an approximately 7 kb insert derived from the 5'10680 bp of Ad2 inserted between the Clal and BamHI sites of pBR322.From this Ad2 fragment, the sequences corresponding to Ad2 nucleotides546-3497 were deleted and replaced with a 12 bp multiple cloning sitecontaining an Nhel site, an Mlul site, and a SnaBI site. The constructalso contains the 5' inverted terminal repeat and viral packagingsignals, the E1a enhancer and promoter, the E1b 3' intron and the 3'untranslated region and polyadenylation sites. The resulting plasmid wascalled pBR-Ad2-7/CFTR. Its use to assemble virus is described below.

2. Virus Preparation from DNA--To generate the recombinant Ad2/CFTR-ladenovirus, the vector pBR-Ad2-7/CFTR was cleaved with BstB1 at the sitecorresponding to the unique BstB1 site at 10670 in Ad2. The cleavedplamid DNA was ligated to BstB1 restricted Ad2 DNA. Following ligation,the reaction was used to transfect 293 cells by the calcium phosphateprocedure. Approximately 7-8 days following transfection, a singleplaque appeared and was used to reinfect a dish of 293 cells. Followingdevelopment of cytopathic effect (CPE), the medium was removed andsaved. Total DNA was prepared from the infected cells and analyzed byrestriction analysis with multiple enzymes to verify the integrity ofthe construct. Viral supernatant was then used to infect 293 cells andupon delvelopment of CPE, expression of CFTR was assayed by the proteinkinase A (PKA) immunoprecipitation assay (Gregory, R. J. et al. (1990)Nature 347:382). Following these verification procedures, the virus wasfurther purified by two rounds of plaque purification.

Plaque purified virus was grown into a small seed stock by inoculationat low multiplicities of infection onto 293 cells grown in monolayers in925 medium supplemented with 10% bovine calf serum. Material at thisstage was designated a Research Viral Seed Stock (RVSS) and was used inall preliminary experiments.

3. Virus Host Cell--Ad2/CFTR-1 is propagated in human 293 cells (ATCCCRL 1573). These cells are a human embryonal kidney cell line which wereimmortalized with sheared fragments of human Ad5 DNA. The 293 cell lineexpresses adenovirus early region 1 gene products and in consequence,will support the growth of E1 deficient adenoviruses. By analogy withretroviruses, 293 cells could be considered a packaging cell line, butthey differ from usual retrovirus lines in that they do not providemissing viral structural proteins, rather, they provide only somemissing viral early functions.

Production lots of virus are propagated in 293 cells derived from theWorking Cell Bank (WCB). The WCB is in turn derived from the Master CellBank (MCB) which was grown up from a fresh vial of cells obtained fromATCC. Because 293 cells are of human origin, they are being testedextensively for the presence of biological agents. The MCB and WCB arebeing characterized for identity and the absence of adventitious agentsby Microbiological Associates, Rockville, Md.

4. Growth of Production Lots of Virus

Production lots of Ad2/CFTR-1 are produced by inoculation ofapproximately 5-10×10⁷ pfu of MVSS onto approximately 1-2×10⁷ Wcb 293cells grown in a T175 flask containing 25 mls of 925 medium. Inoculationis achieved by direct addition of the virus (approximately 2-5 mls) toeach flask. Batches of 50-60 flasks constitute a lot.

Following 40-48 hours incubation at 37° C., the cells are shaken loosefrom the flask and transferred with medium to a 250 ml centrifuge bottleand spun at 1000 xg. The cell pellet is resuspended in 4 ml phosphatebuffered saline containing 0.1 g/l CaCl₂ and 0.1 g/l MgCl₂ and the cellssubjected to cycles of freeze-thaw to release virus. Cellular debris isremoved by centrifugation at 1000 xg for 15 min. The supernatant fromthis centrifugation is layered on top of the CsCl step gradient: 2 ml1.4 g/ml CsCl and 3 ml 1.25 g/ml CsCl in 10 mM Tris, 1 mM EDTA (TE) andspun for 1 hour at 35,000 rpm in a Beckman SW41 rotor. Virus is thenremoved from the interface between the two CsCl layers, mixed with 1.35g/ml CsCl in TE and then subjected to a 2.5 hour equilibriumcentrifugation at 75,000 rpm in a TLN-100 rotor. Virus is removed bypuncturing the side of the tube with a hypodermic needle and gentlyremoving the banded virus. To reduce the CsCl concentration, the sampleis dialyzed against 2 changes of 2 liters of phosphate buffered salinewith 10% sucrose.

Following this procedure, dialyzed virus is stable at 4° C. for severalweeks or can be stored for-longer periods at -80° C. Aliquots ofmaterial for human use will be tested and while awaiting the results ofthese tests, the remainder will be stored frozen. The tests to beperformed are described below:

5. Structure and Purity of Virus

SDS polyacrylamide gel electrophoresis of purified virions reveals anumber of polypeptides, many of which have been characterized. Whenpreparations of virus were subjected to one or two additional rounds ofCsCl centrifugation, the protein profile obtained was indistinguishable.This indicates that additional equilibrium centrifugation does notpurify the virus further, and may suggest that even the less intensebands detected in the virus preparations represent minor virioncomponents rather than contaminating proteins. The identity of theprotein bands is presently being established by N-terminal sequenceanalysis.

6. Contaminating Materials--The material to be administered to patientswill be 2×10⁶ pfu, 2×10⁷ pfu and 5×10⁷ pfu of purified Ad2/CFTR-1.Assuming a minimum particle to pfu ratio of 500, this corresponds to1×10⁹, 1×10¹⁰ and 2.5×10¹⁰ viral particles, these correspond to a doseby mass of 0.25 μg, 2.5 μg and 6.25 μg assuming a moleuclar mass foradenovirus of 150×10⁶.

The origin of the materials from which a production lot of the purifiedAd2/CFTR-1 is derived was described in detail above and is illustratedas a flow diagram in FIG. 6. All the starting materials from which thepurified virus is made (i.e., MCB, and WCB, and the MVSS) will beextensively tested. Further, the growth medium used will be tested andthe serum will be from only approved suppliers who will provide testcertificates. In this way, all the components used to generate aproduction lot will have been characterized. Following growth, theproduction lot virus will be purified by two rounds of CsClcentrifugation, dialyzed, and tested. A production lot should constitute1-5×10¹⁰ pfu Ad2/CFTR-1.

As described above, to detect any contaminating material aliquots of theproduction lot will be analyzed by SDS gel electrophoresis andrestriction enzyme mapping. However, these tests have limitedsensitivity. Indeed, unlike the situation for purified single chainrecombinant proteins, it is very difficult to quantitate the purity ofthe AD2/CFTR-1 using SDS polyacrylamide gel electrophoresis (or similarmethods). An alternative is the immunological detection of contaminatingproteins (IDCP). Such an assay utilizes antibodies raised against theproteins purified in a mock purification run. Development of such anassay has not yet been attempted for the CsCl purification scheme forAd2/CFTR-1. However, initially an IDCP assay developed for the detectionof contaminants in recombinant proteins produced in Chinese hamsterovary (CHO) cells will be used. In addition, to hamster proteins, theseassays detect bovine serum albumin (BSA), transferrin and IgG heavy andlight chain derived from the serum added to the growth medium. Testsusing such reagents to examine research batches of Ad2/CFTR-1 by bothELISA and Western blots are in progress.

Other proteins contaminating the virus preparation are likely to be fromthe 293 cells--that is, of human origin. Human proteins contaminatingtherapeutic agents derived from human sources are usually notproblematic. In this case, however, we plan to test the production lotfor transforming factors. Such factors could be activities ofcontaminating human proteins or of the Ad2/CFR-1 vector or othercontaminating agents. For the test, it is proposed that 10 dishes of RatI cells containing 2×10⁶ cells (the number of target cells in thepatient) with 4 times the highest human dose of Ad2/CFTR-1 (2×10⁸ pfu)will be infected. Following infection, the cells will be plated out inagar and examined for the appearance of transformed foci for 2 weeks.Wild type adenovirus will be used as a control.

Nucleic acids and proteins would be expected to be separated frompurified virus preparations upon equilibrium density centrifugation.Furthermore, the 293 cells are not expected to contain VL30 sequences.Biologically active nucleic cells should be detected.

Example 8

Preliminary Experiments Testing the Ability of Ad2/Gal Virus to EnterAirway Epithelial Cells

a. Hamster Studies

Initial studies involving the intratracheal instillation of the Ad-βGalviral vector into Syrian hamsters, which are reported to be permissivefor human adenovirus are being performed. The first study, a time courseassessment of the pulmonary and systemic acute inflammatory response toa single intratracheal administration of Ad-βGal viral vector, has beencompleted. In this study, a total of 24 animals distributed among threetreatment groups, specifically, 8 vehicle control, 8 low dose virus(1×10¹¹ particles; 3×10⁸ pfu), and 8 high dose virus (1.7×10¹²particles; 5×10⁹ pfu), were used. Within each treatment group, 2 animalswere analyzed at each of four time points after viral vectorinstillation: 6 hrs, 24 hrs, 48 hrs, and 7 days. At the time ofsacrifice of each animal, lung lavage and blood samples were taken foranalysis. The lungs were fixed and processed for normal light-levelhistology. Blood and lavage fluid were evaluated for total leukocytecount and leukocyte differential. As an additional measure of theinflammatory process, lavage fluid was also evaluated for total protein.Following embeddings, sectioning and hematoxylin/eosin staining, lungsections were evaluated for signs of inflammation and airway epithelialdamage.

With the small sample size, the data from this preliminary study werenot amenable to statistical analyses, however, some general trends couldbe ascertained. In the peripheral blood samples, total leukocyte countsshowed no apparent dose- or time- dependent changes. In the bloodleukocyte differential counts, there may have been a minor dose-relatedelevation in percent neutrophil at 6 hours; however, data from all othertime points showed no elevation in neutrophil percentages. Takentogether, these data suggest little or nor systemic inflammatoryresponse to the viral administration.

From the lung lavage, some elevation in total neutrophil counts wereobserved at the first three time points (6 hr, 24 hr, 48 hr). By sevendays, both total and percent neutrophil values had returned to normalrange. The trends in lung lavage protein levels were more difficult toassess due to inter-animal variability; however, no obvious dose- ortime-dependent effects were apparent. First, no damage to airwayepithelium was observed at any time point or virus dose level. Second, atime- and dose- dependent mild inflammatory response was observed, beingmaximal at 48 hr in the high virus dose animals. By seven days, theinflammatory response had completely resolved, such that the lungs fromanimals in all treatment groups were indistinguishable.

In summary, a mild, transient, pulmonary inflammatory response appearsto be associated with the intratracheal administration of the describeddoses of adenoviral vector in the Syrian Hamster.

A second, single intratracheal dose, hamster study has been initiated.This study is designed to assess the possibility of the spread ofineffective viral vectors to organs outside of the lung and the antibodyresponse of the animals to the adenoviral vector. In this study, thethree treatment groups (vehicle control, low dose virus, high dosevirus) each contained 12 animals. Animals will be evaluated at threetime points: I day, 7 days, and 1 month. In this study, viral vectorpersistence and possible spread will be evaluated by the assessment ofthe presence of infective virions in numerous organs including lung,gut, heart, liver, spleen, kidney, brain and gonads. Changes inadenoviral antibody titer will be measured in peripheral blood and lunglavage. Additionally, lung lavage, peripheral blood and lung histologywill be evaluated as in the previous study.

b. Primate studies

Studies of recombinant adenovirus are also underway in primates. Thegoal of these studies is to assess the ability of recombinant adenoviralvectors to deliver genes to the respiratory epithelium in vivo and toassess the safety of the construct in primates. Initial studies inprimates targeted nasal epithelia as the site of infection because ofits similarity to lower airway epithelia, because of its accessibility,and because nasal epithelia was used for the first human studies. TheRhesus monkey (Macaca mulatta) has been chosen for studies, because ithas a nasal epithelium similar to that of humans.

How expression of CFTR affects the electrolyte transport properties ofthe nasal epithelium can be studied in patients with cystic fibrosis.But because the primates have normal CFTR function, instead the abilityto transfer a reporter gene was assessed. Therefore the Ad--βGal viruswas used. The epithelial cell density in the nasal cavity of the Rhesusmonkey is estimated to be 2×10⁶ cells/cm (based on an average nasalepithelial cell diameter of 7 μm) and the surface near 25-50 cm². Thus,there are about 5×10⁷ cells in the nasal epithelium of Rhesus monkey. Tofocus especially on safety, the higher viral doses (20-200 MOI) wereused in vivo. Thus doses in the range of 10⁹ -10¹⁰ pfu were used.

In the first pilot study the right nostril of Monkey A was infected withAd-β-Gal (˜1 ml). This viral preparation was purified by CsCl gradientcentrifugation and then by gel filtration chromatography one week later.Adenoviruses are typically stable in CsCl at 4° C. for one to two weeks.However, this viral preparation was found to be defective (i.e., it didnot produce detectable β-galactosidase activity in the permissive 293cells). Thus, it was concluded that there was no live viral activity inthe material. β-galactosidase activity in nasal epithelial cells fromMonkey A was also not detected. Therefore, in the next study, twodifferent preparations of Ad-β-Gal virus: one that was purified on aCsCl gradient and then dialyzed against Tris-buffered saline to removethe CsCl, and a crude unpurified one was used. Titers of Ad-β-Galviruses were ˜2×10¹⁰ pfu/ml and >1×10¹³ pfu/ml, respectively, and bothpreparations produced detectable β-galactosidase activity in 293 cells.

Monkeys were anesthetized by intramuscular injection of ketamine (15mg/kg). One week before administration of virus, the nasal mucosa ofeach monkey was brushed to establish baseline cell differentials andlevels of β-galactosidase. Blood was drawn for baseline determination ofcell differentials, blood chemistries, adenovirus antibody titers, andviral cultures. Each monkey was also examined for weight, temperature,appetite, and general health prior to infection.

The entire epithelium of one nasal cavity was used in each monkey. Afoley catheter (size 10) was inserted through each nasal cavity into thepharynx, inflated with 2-3 ml of air. and then pulled anteriorly toobtain tight posterior occlusion at the posterior choana. Both nasalcavities were then irrigated with a solution (˜5 ml) of 5 mMdithiothreitol plus 0.2 U/ml neuraminidase in phosphate-buffered saline(PBS) for five minutes. This solution was used to dissolve any residualmucus overlaying the epithelia (It was subsequently found that suchtreatment is not required.) The washing procedure also allowed thedetermination of whether the balloons were effectively isolating thenasal cavity. The virus (Ad-β-Gal) was then slowly instilled into theright nostril with the posterior balloon inflated. The viral solutionremained in contact with the nasal mucosa for 30 minutes. At the end of30 minutes, the remaining viral solution was removed by suction. Theballoons were deflated, the catheters removed, and the monkey allowed torecover from anesthesia. Monkey A received the CsCl-purified virus (˜1.5ml) and Monkey B received the crude virus (˜6 ml). (note that this wasthe second exposure of Monkey A to the recombinant adenovirus).

Both monkeys were followed daily for appearance of the nasal mucosa,conjunctivitis, appetite, activity, and stool consistency. Each monkeywas subsequently anesthetized on days 1, 4, 7, 14, and 21 to obtainnasal, pharyngeal, and tracheal cell samples (either by swabs orbrushes) as described below. Phlebotomy was performed over the same timecourse for hematology, ESR, general screen, antibody serology and viralcultures. Stools were collected every week to assess viral cultures.

To obtain nasal epithelial cells from an anesthetized monkey, the nasalmucosa was first impregnated with 5 drops of Afrin (0.05% oxymetazolinehydrochloride, Schering-Plough) and 1 ml of 2% Lidocaine for 5 min. Acytobrush (the kind typically used for Pap smears) was then used togently rub the mucosa for about 10 seconds. For tracheal brushings, aflexible fiberoptic bronchoscope; a 3 mm cytology brush (Bard) wasadvanced through the bronchoscope into the trachea, and a small area wasbrushed for about 10 seconds. This procedure was repeated twice toobtain a total of ˜10⁶ cells/ml. Cells were then collected on slides(approximately 2×10⁴ cells/slide using a Cytospin 3 (Shandon, Pa.)) forsubsequent staining (see below).

To determine viral efficacy, nasal, pharyngeal, and tracheal cells werestained for β-galactosidase using X-gal (5bromo-4chloro-3-indolyl-β-D-galactoside). Cleavage of X-gal byβ-galactosidase produces a blue color that can be seen with lightmicroscopy. The Ad-β-gal vector included a nuclear-localization signal(NLS) (from SV40 large T-antigen) at the amino-terminus of theβ-galactosidase sequence to direct expression of this protein to thenucleus. Thus, the number of blue nuclei after staining was determined.

RT-PCR (reverse transcriptase-polymerase chain reaction) was also usedto determine viral efficacy. This assay indicates the presence ofβ-galactosidase mRNA in cells obtained by brushings or swabs. PCRprimers were used in both the adenovirus sequence and the LacZ sequenceto distinguish virally-produced mRNA from endogenous mRNA. PCR was alsoused to detect the presence of the recombinant adenovirus DNA. Cytospinpreparations was used to assess for the presence of virally producedβ-galactosidase mRNA in the respiratory epithelial cells using in-situhybridization. This technique has the advantage of being highly specificand will allow assessment which cells are producing the mRNA.

Whether there was any inflammatory response was assessed by visualinspection of the nasal epithelium and by cytological examination ofWright-stained cells (cytospin). The percentage of neutrophils andlymphocytes were compared to that of the control nostril and to thenormal values from, four control monkeys. Systemic repsonses by whiteblood cell counts, sedimentation rate, and fever were also assessed.

Viral replication at each of the time points was assessed by testing forthe presence of live virus in the supernatant of the cell suspensionfrom swabs or brushes. Each supernatant was used to infect (at severaldilutions) the virus-sensitive 293 cell line. Cytopathic changes in the293 cells were monitored for 1 week and then the cells were fixed andstained for β-galactosidase. Cytopathic effects and blue-stained cellsindicated the presence of live virus. Positive supernatants will also besubjected to analysis of nonintegrating DNA to identify (confirm) thecontributing virus(es).

Antibody titers to type 2 adenovirus and to the recombinant adenoviruswere determined by ELISA. Blood/serum analysis was performed using anautomated chemistry analyzer Hitachi 737 and an automated hematologyanalyzer Technicom H6. The blood buffy coat was cultured in A549 cellsfor wild type adenovirus and was cultured in the permissive 293 cells.

Results: Both monkeys tolerated the procedure well. Daily examinationrevealed no evidence of coryza, conjunctivitis or diarrhea For bothmonkeys, the nasal mucosa was mildly erythematous in both the infectionside and the control side; this was interpreted as being due to theinstrumentation. Appetites and weights were not affected by virusadministrated in either monkey. Physical examination on days 1, 4,7, 14and 21 revealed no evidence of lymphadenopathy, tachypnea, ortachycardia. On day 21, monkey B had a temperature 39.1° C. (normal forRhesus monkey 38.8° C.) but had no other abnormalities on physical examor in laboratory data. Monkey A had a slight leukocytosis on day 1 postinfection which returned to normal by day 4; the WBC was 4,920 on theday of infection, 8,070 on day 1, and 5,200 on day 4. The ESR did notchange after the infection. Electrolytes and transaminases were normalthroughout.

Wright stains of cells from nasal brushing were performed on days 4, 7,14, and 21. They revealed less than 5% neutrophils and lymphocytes.There was no difference between the infected and the control side.

X-Gal stains of the pharyngeal swabs revealed blue-stained cells in bothmonkeys on days 4, 7, and 14; only a few of the cells had clear nuclearlocalization of the pigment and some pigment was seen in extracellulardebris. On day 7 post infection, X-Gal stains from the right nostril ofmonkey A, revealed a total of 135 ciliated cells with nuclear-localizedblue stain. The control side had only 4 blue cells Monkey B had 2 bluecells from the infected nostril and none from the control side. Bluecells were not seen on day 7, 14, or 21.

RT-PCR on day 3 post infection revealed a band of the correct size thathybridized with a β-Gal probe, consistent with β-Gal mRNA in the samplesfrom Monkey A control nostril and Monkey B infected nostril. On day 7there was a positive band in the sample from the infected nostril ofMonkey A, the same specimen that revealed blue cells.

Fluid from each nostril, the pharynx, and trachea of both monkeys wasplaced on 293 cells to check for the presence of live virus bycytopathic effect and X-Gal stain. In Monkey A, live virus was detectedin both nostrils on day 3 after infection; no live virus was detected ateither one or two weeks post-infection. In Monkey B, live virus wasdetected in both nostrils, pharynx, and trachea on day 3, and only inthe infected nostril on day 7 after infection. No live virus wasdetected 2 weeks after the infection.

c. Human Explant Studies

In a second type of experiment, epithelial cells from a nasal polyp of aCF patient were cultured on permeable filter supports. These cells forman electrically tight epithelial monolayer after several days inculture. Eight days after seeding, the cells were exposed to theAd2/CFTR virus for 6 hours. Three days later, the short-circuit current(1 sc) across the monolayer was measured. cAMP agonists did not increasethe 1 sc, indicating that there was no change in chloride secretion.However, this defect was corrected after infection with recombinantAd2/CFTR. Cells infected with Ad2/CFTR (MOI=5; MOI refers tomultiplicity of infection; 1 MOI indicates one pfu/cell) expressfunctional CFTR; cAMP agonists stimulated 1 sc, indicating stimulationof Cl⁻ secretion. Ad2/CFTR also corrected the CF chloride channel defectin CF tracheal epithelial cells. Additional studies indicated thatAd2/CFTR was able to correct the chloride secretory defect withoutaltering the transepithelial electrical resistance; this resultindicates that the integrity of the epithelial cells and the tightjunctions was not disrupted by infection with Ad2/CFTR Application of 1MOI of Ad2/CFTR was also found to be sufficient to correct the CFchloride secretory defect

The experiments using primary cultures of human airway epithelial cellsindicate that the Ad2/CFTR virus is able to enter CF airway epithelialcells and express sufficient CFTR to correct the defect in chloridetransport.

Example 9

In Vivo Delivery to and Expression of CFTR in Cotton Rat and RhesusMonkey Epithelium

MATERIALS AND METHODS

Adenovirus vector

Ad2/CFTR-1 was prepared as described in Example 7. The DNA constructcomprises a full length copy of the Ad2 genome of approximately 37.5 kbfrom which the early region 1 genes (nucleotides 546 to 3497) have beenreplaced by cDNA for CFTR (nucleotides 123 to 4622 of the published CFTRsequence with 53 additional linker nucleotides). The viral E1a promoterwas used for CFTR cDNA. Termination/polyadenylation occurs at the sitenormally used by the E1b and protein IX transcripts. The recombinantvirus E3 region was conserved. The size of the Ad2-CFTR-1 vector isapproximately 104.5% that of wild-type adenovirus. The recombinant viruswas grown in 293 cells that complement the E1 early viral promoters. Thecells were frozen and thawed three times to release the virus and thepreparation was purified on a CsCl gradient, then dialyzed againstTris-buffered saline (PBS) to remove the CsCl, as described.

Animals

Rats. Twenty two cotton rats (68 weeks old, weighing between 80-100 g)were used for this study. Rats were anesthetized by inhaledmethoxyflurane (Pitman Moore, Inc., Mundelen, Ill.). Virus was appliedto the lungs by nasal instillation during inspiration.

Two cotton rat studies were performed. In the first study, seven ratswere assigned to a one time pulmonary infection with 100 μl solutioncontaining 4.1×10⁹ plaque forming units (pfa) of the Ad2/CFTR-1 virusand 3 rats served as controls. One control rat and either two or threeexperimental rats were sacrificed with methoxyflurane and studies ateach of three time points: 4, 11, or 15 days after infection.

The second group of rats was used to test the effect of repeatadministration of the recombinant virus. All 12 rats received 2.1×10⁸pfu of the Ad2/CFTR-1 virus on day 0 and 9 of the rats received a seconddose of 3.2×10⁸ pfu of Ad2/CFTR-1 14 days later. Groups of one controlrat and three experimental rats were sacrificed at 3, 7, or 14 daysafter the second administration of virus. Before necropsy, the tracheawas cannulated and brochoaveolar lavage (BAL) was performed with 3 mlaliquots of phosphate-buffered saline. A median stemotomy was performedand the right ventricle cannulated for blood collection. The right lungand trachea were fixed in 4% formaldehyde and the left lung was frozenin liquid nitrogen and kept at ˜70° C. for evaluation byimmunochemistry, reverse transcriptase polymerase chain reaction(RT-PCR), and viral culture. Other organs were removed and quicklyfrozen in liquid nitrogen for evaluation by polymerase chain reaction(PCR).

Monkeys. Three female Rhesus monkeys were used for this study; a fourthfemale monkey was kept in the same room, and was used as control. Forapplication of the virus, the monkeys were anesthetized by intramuscularinjection of ketamine (15 mg/kg). The entire epithelium of one nasalcavity in each monkey was used for virus application. A foley catheter(size 10) was inserted through each nasal cavity into the pharynx, theballoon was inflated with 2-3 ml of air, and then pulled anteriorly toobtain a tight occlusion at the posterior choana. The Ad2/CFTR-1 viruswas then instilled slowly in the right nostril with the posteriorballoon inflated. The viral solution remained in contact with the nasalmucosa for 30 min. The balloons were deflated, the catheters wereremoved, and the monkeys were allowed to recover from anesthesia. Asimilar procedure was performed on the left nostril, except that TBSsolution was instilled as a control. The monkeys received a total ofthree doses of the virus over a period of 5 months. The total dose givenwas 2.5×10⁹ pfu the first time, 2.3×10⁹ pfu the second time, and 2.8×10⁹pfu the third time. We estimated that the cell density of the nasalepithelia to be 2×10⁶ cells/cm² and a surface area of 25 to 50 cm². Thiscorresponds to a multiplicity of infection (MOI) of approximately 25.

The animals were evaluated 1 week before the first administration ofvirus, on the day of administration, and on days 1, 3, 6, 13, 21, 27,and 42 days after infection. The second administration of virus occurredon day 55. The monkeys were evaluated on day 55 and then on days 56, 59,62, 69, 76, 83, 89, 96, 103, and 111. For the third administration, onday 134, only the left nostril was cannulated and exposed to the virus.The control monkey received instillations of PBS instead of virus.Biopsies of the left medial turbinate were carried out on day 135 in oneof the infected monkeys, on day 138 on the second infected monkey, andon day 142 on the third infected monkey and on the control monkey.

For evaluations, monkeys were anesthetized by intramuscular injection ofketamine (15 mg/kg). To obtain nasal epithelial cells, the nasal mucosawas first impregnated with 5 drops of Afrin (0.05% oxymetazolinehydrochloride, Schering-Plough) and 1 ml of 2% Lidocaine for 5 minutes.A cytobrush was then used to gently rub the mucosa for about 3 sec. Toobtain pharyngeal epithelial swabs, a cotton-tipped applicator wasrubbed over the back of the pharynx 2-3 times. The resulting cells weredislodged from brushes or applicators into 2 ml of sterile PBS. Biopsiesof the medial turbinate were performed using cupped forceps under directendoscopic control.

Animals were evaluated daily for evidence of abnormal behavior ofphysical signs. A record of food and fluid intake was used to assessappetite and general health. Stool consistency was also recorded tocheck for the possibility of diarrhea. At each of the evaluation timepoints, we measured rectal temperature, respiratory rate, and heartrate. We visually inspected the nasal mucosa, conjunctivas, and pharynx.The monkeys were also examined for lymphadenopathy.

Venous blood from the monkeys was collected by standard venipuncturetechnique. Blood/serum analysis was performed in the clinical laboratoryof the University of Iowa Hospitals and Clinics using a Hitachi 737automated chemistry analyzer and a Technicom H6 automated hematologyanalyzer.

Serology

Sera were obtained and anti-adenoviral antibody titers were measured byan enzyme-linked immunoadsorbant assay (ELISA). For the ELISA, 50ng/well of filled adenovirus (Lee Biomolecular Research Laboratories,San Diego, Calif.) in 0.1M NaHCO₃ were coated on 96 well plates at 4° C.overnight. The test samples at appropriate dilutions were added,starting at a dilution of 1/50. The samples were incubated for 1 hour,the plates washed, and a goat anti-human IgG HRP conjugate (JacksonImmunoResearch Laboratories, West Grove, Pa.) was added and incubatedfor 1 hour. The plates were washed and O-Phenylenediamine (SigmaChemical Co., St. Louis, Mo.) was added for 30 min. at room temperature.The assay was stopped with 4.5M H₂ SO₄ and read 490 nm on a MolecularDevices microplate reader. The titer was calculated as the product ofthe reciprocal of the initial dilution and the reciprocal of thedilution in the last well with an OD>0.100.

Neutralizing antibodies measure the ability of the monkey serum toprevent infection of 293 cells by adenovirus. Monkey serum (1:25dilution) or nasal washings (1:2 dilutions)! were added in two-foldserial dilutions to a 96 well plate. Adenovirus (2.5×10⁵ pfu was addedand incubated for 1 hour at 37° C. The 293 cells were then added to allwells and the plates were incubated until the serum-free control wellsexhibited >95% cytopathic effect. The titer was calculated as theproduct of the reciprocal of the initial dilution times the reciprocalof the dilution in the last well showing >95% cytopathic effect.

Bronchoalveolar lavage and nasal brushings for cytology

Bronchoalveolar lavage (BAL) was performed by cannulating the tracheawith a silastic catheter and injecting 5 ml of PBS. Gentle suction wasapplied to recover the fluid. The BAL sample was spun at 5000 rpm for 5min. and cells were resuspended in 293 media at a concentration of 10⁶cells/ml. Cells were obtained from the monkey's nasal epithelium bygently rubbing the nasal mucosa for about 3 sec. with a cytobrush. Theresulting cells were dislodged from the brushes into 2 ml of PBS. Fortymicroliters of the cell suspension were cytocentrifuged onto slides andstained with Wright's stain. Samples were examined by light microscopy.

Histology of lung sections and nasal biopsies

The right lung of each cotton rat was removed, inflated with 4%formaldehyde, and embedded in paraffin for sectioning. Nasal biopsiesfrom the monkeys were also fixed with 4% formaldehyde. Histologicsections were stained with hematoxylin and eosin (H&E). Sections werereviewed by at least one of the study personnel and by a pathologist whowas unaware of the treatment each rat received.

Immunoytochemistry

Pieces of lung and trachea of the cotton rats and nasal biopsies werefrozen in liquid nitrogen on O.C.T. compound. Cryosections and paraffinsections of the specimens were used for immunofluorescence rnicroscopy.Cytospin slides of nasal brushings were prepared on gelatin coatedslides and fixed with paraformaldehyde. The tissue was permeabilizedwith Triton X-100, then a pool of monoclonal antibodies to CFTR (M13-1,M14) (Denning, G. M. et al. (1992) J. Clin. Invest. 89:339-349) wasadded and incubated for 12 hours. The primary antibody was removed andan anti-mouse biotinylated antibody (Biomeda, Foster City, Calif.) wasadded. After removal of the secondary antibody, streptavidin FITC(Biomeda, Foster City, Calif.) was added and the slides were observedunder a laser scanning confocal microscope. Both control animal samplesand non-immune IgG stained samples were used as controls.

PCR

PCR was performed on pieces of small bowel, brain, heart, kidney, liver,ovaries, and spleen from cotton rats. Approximately 1 g of the ratorgans was mechanically ground and mixed with 50 μI sterile water,boiled for 5 min., and centrifuged. A 5 μl aliquot of the supernatantwas removed for further analysis. Monkey nasal brushings suspensionswere also used for PCR.

Nested PCR primer sets were designed to selectively amplify Ad2/CFTR-1DNA over endogenous CFTR by placing one primer from each set in theadenovirus sequence and the other primer in the CFTR sequence. The firstprimer set amplifies a 723 bp fragment and is shown below:

Ad2 5' ACT CTT GAG TGC CAG CGA GTA GAG TTT TCT CCT CCG 3'(SEQ ID NO:4)

CFTR 5' GCA AAG GAG CGA TCC ACA CGA AAT GTG CC 3'(SEQ ID NO:5)

The nested primer set amplifies a 506 bp fragment and is shown below:

Ad2 5' CTC CTC CGA GCC GCT CCG AGC TAG 3'(SEQ ID NO:6)

CFTR 5' CCA AAA ATG GCT GGG TGT AGG AGC AGT GTC C 3'(SEQ ID NO:7)

A PCR reaction mix containing 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mMMgCl₂, 0.001% (w/v) gelatin, 400 μM each dNTP, 0.6 μM each primer (firstset), and 2.5 units AmpliTaq (Perkin Elmer) was aliquoted into separatetubes. A 5 μl aliquot of each sample prep was then added and the mixturewas overlaid with 50 μl of light mineral oil. The samples were processedon a Barnstead/Thermolyne (Dubuque, Iowa) thermal cycler programmed for1 min. at 94° C., 1 min. at 65° C., and 2 min. at 72° C. for 40 cycles.Post-run dwell was for 7 min. at 72° C. A 5 μl aliquot was removed andadded to a second PCR reaction using the nested set of primers andcycled as above. A 10 μl aliquot of the final amplification reaction wasanalyzed on a 1% agarose gel and visualized with ethidium bromide.

To determine the sensitivity of this procedure, a PCR mix containingcontrol rat liver supernatant was aliquoted into several tubes andspiked with dilutions of Ad2/CFTR-1. Following the amplificationprotocols described above, it was determined that the nested PCRprocedure could detect as little as 50 pfu of viral DNA.

RT-PCR

RT-PCR was used to detect vector-generated mRNA in cotton rat lungtissue and samples from nasal brushings from monkeys. A 200 μl aliquotof guanidine isothiocyanate solution (4M quanidine isothiocyanate 25 mMsodium citrate pH 7.0. 0.5% sarcosvl. and 0.1 M β-mercaptoethanol) wasadded to a frozen section of each lung and pellet from nasal brushingsand the tissue was mechanically ground. Total RNA was isolated utilizinga single-step method (Chomczynski, P. and Sacchi, N. et al. (1987)Analytical Biochemistry 162:156-159; Hanson, C. A. et al. (1990) Am. J.Pathol. 137:1-6). The RNA was incubated with 1 unit RQ1 RNase-free DNase(Promega Corp., Madison Wis.)) at 37° C. for 20 min., denatured at 99°C. for 5 min., precipitated with ammonium acetate and ethanol, andredissolved in 4 μl diethylpyrocarbonate treated water containing 20units RNase Block 1 (Stratagene, La Jolla Calif.). A 2 μl aliquot of thepurified RNA was reverse tanscribed using the GeneAmp RNA PCR kit(Perkin Elmer Cetus) and the downstream primer from the first primer setdescribed in the previous section. Reverse transcriptase was omittedfrom the reaction with the remaining 2 μl of the purified RNA prep, as acontrol in which preparations (both +/- RT) were then amplified usingnested primer sets and the PCR protocols described above. A 10 μlaliquot of the final amplification reaction was analyzed on a 1% agarosegel and visualized with ethidium bromide.

Southern analysis

To verify the identity of the PCR products, Southern analysis wasperformed. The DNA was transferred to a nylon membrane as described(Sambrook et al.). A fragment of CFTR cDNA (aminoacids #1-525) waslabeled with ³² P!-dCTP (ICN Biomedicals, Inc. Irvine Calif.) using anoligolabeling kit (Pharmacia, Piscataway, N.J.) and purified over a NICKcolumn (Pharmacia Piscataway, N.J.) for use as a hybridization probe.The labeled probe was denatured, cooled, and incubated with theprehybridized filter for 15 hours at 42° C. The hybridized filter wasthen exposed to film (Kodak XAR-5) for 10 min.

Culture of Ad2/CFTR-1

Viral cultures were performed on the permissive 293 cell line. Forculture of virus from lung tissue, 1 g of lung was frozen/thawed 3-6times and then mechanically disrupted in 200 μl of 293 media For cultureof BAL and monkey nasal brushings, the cell suspension was spun for 5min and the supernatant was collected. Fifty μl of the supernatant wasadded in duplicate to 293 cells grown in 96 well plates at 50%confluence. The 293 cells were incubated for 72 hr at 37° C., then fixedwith a mixture of equal parts of methanol and acetone for 10 min. andincubated with FITC-labeled antiadenovirus monoclonal antibodies(Chemicon, Light Diagnostics, Temecuca, Calif.) for 30 min. Positivenuclear immunofluorescence was interpreted as positive culture. Thesensitivity of the assay was evaluated by adding dilutions of Ad2/CFTR-1to 50 μl of the lung homogenate from one of the control rats. Viralreplication was detected when as little as 1 pfu was added.

RESULTS

Efficacy of Ad2/CFTR-1 in the lungs of cotton rats

To test the ability of Ad2/CFTR-1 to transfer CFTR cDNA to theintrapulmonary airway epithelium, several studies were performed. 4×10pfu--I.U. of Ad2/CFTR-1 in 100 μl s adminstered to seven cotton rats;three control rats received 100 μl of TBS (the vehicle for the virus).The rats were sacrificed 4, 10 or 14 days later. To detect viraltranscripts encoding CFTR, reverse transcriptase was used to preparecDNA from lung homogenates. The cDNA was amplified with PCR usingprimers that span adenovirus and CFTR-encoded sequences. Thus, theprocedure did not detect endogenous rat CFTR. The lungs of animals whichreceived Ad2/CFTR-1 were positive for virally-encoded CFTR mRNA. Thelungs of all control rats were negative.

To detect the protein, lung sections were immunostained with antibodiesspecific to CFTR. CFTR was detected at the apical membrane of bronchialepithelium from all rats exposed to Ad2/CFTR-1, but not from controlrats. The location of recombinant CFTR at the apical membrane isconsistent with the location of endogenous CFTR in human airwayepithelium. Recombinant CFTR was detected above background levelsbecause endogenous levels of CFTR in airway epithelia are very low andthus, difficult to detect by immunocytochemistry (Trapnell, B. et al.(1991) Proc. Natl. Acad. Sci USA 88:6565-6569; Denning, G. M. et al.(1992) J Cell Biol. 118:551-59).

These results show that Ad2/CFTR-1 directs the expression of CFTR mRNAin the lung of the cotton rat and CFTR protein in the intrapulmonaryairways.

Safety of Ad2/CFTR-1 in cotton rats

Because the E1 region of Ad2 is deleted in the Ad2/CFTR-1 virus, thevector was expected to be replication-impaired (Berkner, K. L. (1988)BioTechniques 6:616-629) and that it would be unable to shut off hostcell protein synthesis (Basuss, L. E. et al. (1989) J. Virol.50:202-212). Previous in vitro studies have suggested that this is thecase in a variety of cells including primary cultures of human airwayepithelial cells (Rich, D. P. et al. (1993) Human Gene Therapy4:461-476). However, it is important to confirm this in vivo in thecotton rat, which is the most permissive animal model for humanadenovirus infection (Ginsberg, H. S. et al. (1989) Proc. Natl. Acad.Sci USA 86:3823-3827; Prince, G. A. et al. (1993) J. Virol. 67:101-111).Although dose of virus of 4.1×10¹⁰ pfus per kg was used, none of therats dies. More importantly, extracts from lung homogenates from each ofthe cotton rats were cultured in the permissive 293 cell line. With thisassay 1 pfu of recombinant virus was detected in lung homogenate.However, virus was not detected by culture in the lungs of any of thetreated animals. Thus, the virus did not appear to replicate in vivo.

It is also possible that administration of Ad2/CFTR-1 could cause aninflammatory response, either due to a direct effect of the virus or asa result of administration of viral particles. Several studies wereperformed to test this possibility. None of the rats had a change in thetotal or differential white blood cell count, suggesting that there wasno major systemic inflammatory response. To assess the pulmonaryinflammatory response more directly, bronchoalveolar lavage wasperformed on each of the rats. FIG. 18A shows that there was no changein the total number of cells recovered from the lavage or in thedifferential cell count.

Sections of the lung stained by H&E were also prepared. There was noevidence of viral inclusions or any other changes characteristic ofadenoviral infection (Prince, G. A. et al. (1993) J. Virol. 67:101-111).When coded lung sections were evaluated by a skilled reader who wasunaware of which sections were treated, she was unable to distinguishbetween sections from the treated and untreated lungs.

It seemed possible that the recombinant adenovirus could escape from thelung into other tissues. To test for this possibility, other organs fromthe rats were evaluated using nested PCR to detect viral DNA. All organstested from infected rats were negative, with the exception of smallbowel which was positive in 3 of 7 rats. The presence of viral DNA inthe small bowel suggests that the rats may have swallowed some of thevirus at the time of instillation or, alternatively, the normal airwayclearance mechanisms may have resulted in deposition of viral DNA in thegastrointestinal tract. Despite the presence of viral DNA in homogenatesof small intestine, none of the rats developed diarrhea. This resultsuggests that if the virus expressed CFTR in the intestinal epithelium,there was no obvious adverse consequence.

Repeat administration of Ad2/CFTR-1 to cotton rats

Because adenovirus DNA integration into chromosomal DNA is not necessaryfor gene expression and only occurs at very low frequency, expressionfollowing any given treatments was anticipated to be finite and thatrepeated administration of recombinant adenovirus would be required fortreatment of CF airway disease. Therefore, the effect of repeatedadministration of Ad2/CFTR-1 cotton rats was examined. Twelve cottonrats received 50 μl of Ad2/CFTR-1. Two weeks later, 9 of the ratsreceived a second dose of 50 μl of Ad2/CFTR-1 and 3 rats received 50 μlof TBS. Rats were sacrificed on day 3, 7, or 14 after virusadministration. At the time of the second vector administration allcotton rats had an increased antibody titer to adenovirus.

After the second intrapulmonary administration of virus, none of therats died. Moreover, the results of studies assessing safety andefficacy were similar to results obtained in animals receivingadenovirus for the first time. Viral cultures of rat lung homogenates on293 cells were negative at all time points, suggesting that there was novirus replication. There was no difference between treated and controlrats in the total or differential white blood count at any of the timepoints. The lungs were evaluated by histologic. sections stained withH&E; and found no observable differences between the control and treatedrats when sections were read by us or by a blinded skilled reader. Whenorgans were examined for viral DNA using PCR, viral DNA was found onlyin the small intestine of 2 rats. Despite seropositivity of the rats atthe time of the second administration, expression of CFTR (as assessedby RT-PCR and by immunocytochemistry of sections stained with CFTRantibodies) similar to that seen in animals that received a singleadministration was observed.

These results suggest that prior administration of Ad2/CFTR-1 and thedevelopment of an antibody response did not cause an inflammatoryresponse in the rats nor did it prevent virus-dependent production ofCFTR.

Evidence that Ad2/CFTR-1 expresses CFTR in primate airway epithelium

The cells lining the respiratory tract and the immune system of primatesare similar to those of humans. To test the ability of Ad2/CFTR-1 totransfer CFTR to the respiratory epithelium of primates, Ad2/CFTR wasapplied on three occasions as described in the methods to the nasalepithelium of three Rhesus monkeys. To obtain cells from the respiratoryepithelium, the epithelium was brushed using a procedure similar to thatused to sample the airway epithelium of humans during fiberopticbronchoscopy.

To assess gene transfer, RT-PCR was used as described above for thecotton rats. RT --PCR was positive on cells brushed from the rightnostril of all three monkeys, although it was only detectable for 18days after virus administration. An example of the results are shown inFIG. 19A. The presence of a positive reaction in cells from the leftnostril most likely represents some virus movement to the left side dueto drainage, or possibly from the monkey moving the virus from onenostril to the other with its fingers after it recovered fromanesthesia.

The specificity of the RT-PCR is shown in FIG. 23B. A Southern blot witha probe to CFTR hybridized with the RT-PCR product from the monkeyinfected with Ad2/CFTR-1. As a control, one monkey received a differentvirus (Ad2/βGal-1) which encodes β-galactosidase. When different primerswere used to reverse transcribe the β-galactosidase mRNA and amplify thecDNA, the appropriate PCR product was detected. However, the PCR productdid not hybridize to the CFTR probe on Southern blot. This result showsthe specificity of the reaction for amplification of theadenovirus-directed CFTR transcript.

The failure to detect evidence of adenovirus-encoded CFTR mRNA at 18days or beyond suggests that the sensitivity of the RT-PCR may be lowbecause of limited efficacy of the reverse transcriptase or becauseRNAses may have degraded RNA after cell acquisition. Viral DNA, however,was detected by PCR in brushings from the nasal epithelium for seventydays after application of the virus. This result indicates that althoughmRNA was not detected after 2 weeks, viral DNA was present for aprolonged period and may have been transcriptionally active.

To assess the presence of CFTR proteins directly, cells obtained bybrushing were plated onto slides by cytospin and stained with antibodiesto CFTR. A positive reaction was clearly evident in cells exposed toAd2/CFTR-1. The cells were scored as positive by immunocytochemistrywhen evaluated by a reader uninformed to the identity of the samples.Immunocytochemistry remained positive for five to six weeks for thethree monkeys, even after the second administration of Ad2/CFTR-1. Onoccasion, a few positive staining cells were observed from thecontralateral nostril of the monkeys. However, this was of shortduration, lasting at most one week.

Sections of nasal turbinate biopsies obtained within a week after thethird infection were also examined. In sections from the control monkey,little if any immunofluorescence from the surface epithelium wasobserved, but the submucosal glands showed significant staining of CFTR.These observations are consistent with results of previous studies(Engelhardt, J. F. and Wilson, J. M. (1992) Nature Gen. 2:240-248.) Incontrast, sections from monkeys that received Ad2/CFTR-1 revealedincreased immunofluorescence at the apical membrane of the surfaceepithelium. The submucosal glands did not appear to have greaterimmunostraining than was observed under control conditions. Theseresults indicate that Ad2/CFTR-1 can transfer the CFTR cDNA to theairway epithelium of Rhesus monkeys, even in seropositive animals (seebelow).

Safety of Ad2/CFTR-1 administered to monkeys

FIG. 20 shows that all three treated monkeys developed antibodiesagainst adenovirus. Antibody titers measured by ELISA rose within twoweeks after the first infection. With subsequent infections the titerrose within days. The sentinel monkey had low antibody titers throughoutthe experiment. Tests for the presence of neutralizing antibodies werealso performed. After the first administration, neutralizing antibodieswere not observed, but they were detected after the secondadministration and during the third viral administration (FIG. 20).

To detect virus, supernatants from nasal brushings and swabs werecultured on 293 cells. All monkeys had positive cultures on day 1 and onday 3 or 4 from the infected nostril. Cultures remained positive in oneof the monkeys at seven days after administration, but cultures werenever positive beyond 7 days. Live virus was occasionally detected inswabs from the contra lateral nostril during the first 4 days afterinfection. The rapid loss of detectable virus suggests that there wasnot viral replication. Stools were routinely cultured, but virus wasnever detected in stools from any of the monkeys.

None of the monkeys developed any clinical signs of viral infection orinflammation. Visual inspection of the nasal epithelium revealed slighterythema in all three monkeys in both nostrils on the first day afterinfection; but similar erythema was observed in the control monkey andlikely resulted from the instrumention. There was no visibleabnormalities at days 3 or 4, or on weekly inspection thereafter.Physical examination revealed no fever, lymphadenopathy, conjunctivitis,tachypnea, or tachycardia at any of the time points. No abnormalitieswere found in a complete blood count or sedimentation rate, nor wereabnormalities observed in serum electrolytes, transaminases, or bloodurea nitrogen and creatinine.

Examination of Wright-stained cells from the nasal brushings showed thatneutrophils and lymphocytes accounted for less than 5% of total cells inall three monkeys. Administration of the Ad2/CFTR-1 caused no change inthe distribution or number of inflammatory cells at any of the timepoints following virus administration. H&E stains of the nasal turbinatebiopsies specimens from the control monkey could not be differentiatedfrom that of the experimental monkey when the specimens were reviewed byan independent pathologist.

These results demonstrate the ability of a recombinant adenovirusencoding CFTR (Ad2/CFTR-1) to express CFTR cDNA in the airway epitheliumof cotton rats and monkeys during repeated administration. They alsoindicate that application of the virus involves little if any risk.Thus, they suggest that such a vector may be of value in expressing CFTRin the airway epithelium of humans with cystic fibrosis.

Two methods were used to show that Ad2/CFTR-1 expresses CFTR in theairway epithelium of cotton rats and primates: CFTR mRNA was detectedusing RT-PCR and protein was detected by immunocytochemistry. Durationof expression as assessed immunocytochemically was five to six weeks.Because very little protein is required to generate Cl⁻ secretion(Welsh, M. J. (1987) Physiol. Rev. 67:1143-1184; Trapnell, B. C. et al.(1991) Proc. Natl. Acad Sci. USA 88:6565-6569; Denning, G. M. et al.(1992) J. Cell Biol. 118:551-559), it is likely that functionalexpression of CFTR persists substantially longer than the period of timeduring which CFTR was detected by immunocytochemistry. Support for thisevidence comes from two consderations: first, it is very difficult todetect CFTR immuncytochemically in the airway epithelium, yet theexpression of an apical membrane Cl⁻ permeability due to the presence ofCFTR Cl⁻ channels is readily detected. The ability of a minimal amountof CFTR to have important functional effects is likely a result of thefact that a single ion channel conducts a very large number of ions (10⁶-10⁷ ions/sec). Thus, ion channels are not usually abundant proteins inepithelia Second, previous work suggests that the defective electrolytetransport of CF epithelia can be corrected when only 6-10% of cells in aCF airway epithelium overexpress wild-type CFTR (Johnson, L. G. et al.(1992) Nature Gene 2:21-25). Thus, correction of the biologic defect inCF patients may be possible when only a small percent of the cellsexpress CFTR. This is also consistent with our previous studies in vitroshowing that Ad2/CFTR-1 at relatively low multiplicities of infectiongenerated a cAMP-stimulated Cl⁻ secretory response in CF epithelia(Rich, D. P. et al. (1993) Human Gene Therapy 4:461-476).

This study also provides the first comprehensive data on the safety ofadenovirus vectors for gene transfer to airway epithelium.--Severalaspects of the studies are encouraging. There was no evidence of viralreplication, rather infectious viral particles were rapidly cleared fromboth cotton rats and primates. These data, together with our previous invitro studies, suggest that replication of recombinant virus in humanswill likely not be a problem. The other major consideration for safetyof an adenovirus vector in the treatment of CF is the possibility of aninflammatory response. The data indicate that the virus generated anantibody response in both cotton rats and monkeys. Despite this, noevidence of a systemic or local inflammatory response was observed. Thecells obtained by bronchoalveolar lavage and by brushing and swabs werenot altered by virus application. Moreover, the histology of epitheliatreated with adenovirus was indistinguishable from that of controlepithelia. These data suggest that at least three sequential exposuresof airway epithelium to adenovirus does not cause a detrimentalinflammatory response.

These data suggest that Ad2/CFTR-1 can effectively transfer CFTR cDNA toairway epithelium and direct the expression of CFTR. They also suggestthat transfer is relatively safe in animals. Thus, they suggest thatAd2/CFTR-1 may be a good vector for treating patients with CF. This wasconfirmed in the following example.

Example 10

CFTR Gene Therapy in Nasal Epithelia from Human CF Subjects

EXPERIMENTAL PROCEDURES

Adenovirus vector. The recombinant adenovirus Ad2/CFTR-1 was used todeliver CFTR cDNA. The construction and preparation of Ad2/CFTR-1, andits use in vitro and in vivo in animals, has been previously described(Rich, D. P. et al. (1993) Human Gene Therapy 4:461-476; Zabner, J. etal. (1993) Nature Gen. (in press)). The DNA construct comprises a fulllength copy of the Ad2 genome from which the early region 1 genes(nucleotides 546 to 3497) have been replaced by cDNA for CFTR. The viralE1a promoter was used for CFTR cDNA; this is a low to moderate strengthpromoter. Termination/polyadenylation occurs at the site normally usedby E1b and protein IX transcripts. The E3 region of the virus wasconserved.

Patients

Three patients with CF were studied. Genotype was determined by IG Labs(Framingham, Mass.). All three patients had mild CF as defined by an NIHscore >70 (Taussig, L. M. et al. (1973) J Pediatr. 82:380-390), a normalweight for height ratio, a forced expiratory volume in one second (FEV1)greater than 50% of predicted and an arterial PO₂ greater than 72. Allpatients were seropositive for type 2 adenovirus, and had no recentviral illnesses. Pretreatment cultures of nasal swabs, pharyngeal swabs,sputum, urine, stool, and blood leukocytes were negative for adenovirus.PCR of pretreatment nasal brushings using primers for the adenovirus E1region were negative. Patients were evaluated at least twice by FEV1,cytology of nasal mucosa, visual inspection, and measurement of Vtbefore treatment. Prior to treatment, a coronal computed tomographicscan of the paranasal sinuses and a chest X-ray were obtained.

The first patient was a 21 year old woman who was diagnosed at 3 monthsafter birth. She had pancreatic insufficiency, a positive sweat chloridetest (101 mEq/1), and is homoygous for the ΔF508 mutation. Her NIH scorewas 90 and her FEV1 was 83% predicted. The second patient is a 36 yearold man who was diagnosed at the age of 13 when he presented withsymptoms of pancreatic insufficiency. A sweat chloride test revealed achloride concentration of 70 mEq/1. He is a heteroygote with the ΔF508and G55ID mutations. His NIH score was 88 and his FEV1 was 66%predicted. The third patient is a 50 year old woman, diagnosed at theage of 9 with a positive sweat chloride test (104 mEq/1). She haspancreatic insufficiency and insulin dependent diabetes mellitus. She ishomozygous for the ΔF508 mutation. Her NIH score was 73 and her FEV1 was65% predicted.

Transepithelial voltage

The transepithelial electric potential difference across the nasalepithelium was measured using techniques similar to those previouslydescribed (Alton, E. W. F. W. et al (1987) Thorax 42:815-817; Knowles,M. et al. (1981) N. Eng. J Med. 305:1489-1495). A 23 gauge subcutaneousneedle connected with sterile normal saline solution to a silver/silverchloride pellet (E. W. Wright, Guilford, Conn.) was used as a referenceelectrode. The exploring electrode was a size 8 rubber catheter(modified Argyle^(R) Foley catheter, St. Louis, Mo.) with one side holeat the tip. The catheter was filled with Ringer's solution containing(in mM), 135 NaCl, 2.4 KH₂ PO₂, K₂ HPO₄, 1.2CaCL₂, 1.2 MgCI₂ and 10Hepes (titrated to pH 7.4 with NaOH) and was connected to asilver/silver chloride pellet. Voltage was measured with a voltmeter(Keithley Instruments Inc., Cleveland, Ohio) connected to a strip chartrecorder (Servocorder, Watanabe Instruments, Japan). Prior to themeasurements, the silver/silver chloride pellets were connected inseries with the Ringer's solution; the pellets were changed if therecorded Vt was greater than ±4 mV. The rubber catheter was introducedinto the nostril under telescopic guidance (Hopkins Telescope, KarlStorz, Tuttlingen West Germany) and the side hole of the catheter wasplaced next to the study area in the medical aspect of the inferiornasal turbinate. The distance from the anterior tip of the inferiorturbinate and the spatial relationship with the medial turbinate, themaxillary sinus ostium, and in one patient a small polyp, were used tolocate the area of Ad2/CFTR-1 administration for measurements;Photographs and video recorder images were also used. Basal Vt wasrecorded until no changes in Vt were observed after slow intermittent100 μl/min infusion of the Ringer's solution. Once a stable baseline wasachieved, 200 μl of a Ringer's solution containing 100 μM amiloride(Merck and Co. Inc., West Point, Pa.) was instilled through the catheterand changes in Vt were recorded until no further change were observedafter intermittent instillations. Finally, 200 μI Ringer's solutioncontaining 100 μM amiloride plus 10 μM terbutaline (GeigyPharmaceuticals, Ardsley, N.Y.) was instilled and the changes in Vt wererecorded.

Measurements of basal Vt were reproducible over time: in the threetreated patients, the coefficients of variation before administration ofAd2/CFTR-1 were 3.6%, 12%, and 12%. The changes induced by terbutalinewere also reproducible. In 30 measurements in 9 CF patients, theterbutaline-induced changes in Vt (ΔVt) ranged from 0 mV to +4 mV;hyperpolarization of Vt was never observed. In contrast, in 7 normalsubjects ΔVt ranged from -1 mV to -5 mV; hyperpolarization was alwaysobserved.

Ad2/CFTR-1 application and cell acquisition.

The patients were taken to the operating room and monitoring wascommenced using continuous EKG and pulse oximetry recording as well asautomatic intermittent blood pressure measurement. After mild sedation,the nasal mucosa was anesthetized by atomizing 0.5 ml of 5% cocaine. Themucosa in the area of the inferior turbinate was then packed with cottonpledgets previously soaked in a mixture of 2 ml of 0.1% adrenaline and 8ml of 1% tetracaine. The pledgets remained in place for 1040 min. Usingendoscopic visualization with a television monitoring system, theapplicator was introduced through the nostril and positioned on themedial aspect of the inferior turbinate, at least three centimeters fromits anterior tip (FIGS. 21A-21I). The viral suspension was infused intothe applicator through connecting catheters. The position of theapplicator was monitored endoscopically to ensure that it did not moveand that enough pressure was applied to prevent leakage. After the viruswas in contact with the nasal epithelium for thirty minutes, the viralsuspension was removed, and the applicator was withdrawn. In the thirdpatient's right nasal cavity, the virus was applied using the modifiedFoley catheter used for Vt measurements. The catheter was introducedwithout anesthetic under endoscopic guidance until the side hole of thecatheter was in contact with the area of interest in the inferiorturbinate. The viral solution was infused slowly until a drop ofsolution was seen with the telescope. The catheter was left in place forthirty minutes and then removed.

Cells were obtained from the area of virus administration approximately2 weeks before treatment and then at weekly intervals after treatment.The inferior turbinate was packed for 10 minutes with cotton pledgetspreviously soaked in 1 ml of 5% cocaine. Under endoscopic control, thearea of administration was gently brushed for 5 seconds. The brushedcells were dislodged in PBS. Swabs of the nasal epithelia were collectedusing cotton tipped applicators without anesthesia. Cytospin slides wereprepared and stained with Wright's--stain. Light microscopy was used toassess the respiratory epithelial cells and inflammatory cells. Forbiopsies, sedatives/anesthesia was administered as described for theapplication prodcure. After endosconic inspection and identification ofthe site to be biopsied the submucosa was injected with 1% xylocaine,with 1/100,000 epinephrine. The area of virus application on theinferior turbinate was removed. The specimen was fixed in 4%formaldehyde and stained.

RESULTS

On day one after Ad2/CFTR-1 administration and at all subsequent timepoints, Ad2/CFTR-1 from the nasal epithelium, pharynx, blood, urine, orstool could not be cultured. As a control for the sensitivity of theculture assay, samples were routinely spiked with 10 and 100 I.U.Ad2/CFTR- 1. In every case, the spiked samples were positive, indicatingthat, at a minimum, 10 I.U. of Ad/CFTR should have been detected. Noevidence of a systemic response as assessed by history, physicalexamination, serum chemistries or cell counts, chest and sinus X-rays,pulmonary function tests, or arterial blood gases performed before andafter Ad2/CFTR-1 administration. An increase in antibodies to adenoviruswas not detectable by ELISA or by neutralization for 35 days aftertreatment.

Three to four hours after Ad2/CFTR-1 administration, at the time thatlocal anesthesia and localized vasoconstriction abated, all patientsbegan to complain of nasal congestion and in one case, mild rhinorrhea.These were isolated symptoms that diminished by 18 hours and resolved by28 to 42 hours. Inspection of the nasal mucosa showed mild to moderateerythema, edema, and exudate (FIGS. 21A-21C). These physical findingsfollowed a time course similar to the symptoms. The physical findingswere not limited to the site of virus application, even thoughpreliminary studies using the applicator showed that marker methyleneblue was limited to the area of application. In two additional patientswith CF, the identical anesthesia and application procedure were used,but saline was applied instead of virus, yet the same symptoms andphysical findings were observed in these patients (FIGS. 21G-21I).Moreover, the local anesthesia and vasoconstriction generated similarchanges even when the applicator was not used, suggesting that theanesthesia/vasoconstriction caused some, if not all the injury.Twenty-four hours after the application procedure, analysis of cellsremoved from nasal swabs revealed an equivalent increase in the percentneutrophils in patients treated with Ad2/CFTR-1 or with saline. One weekafter application, the neutrophilia had resolved in both groups.Respiratory epithelial cells obtained by nasal brushing appeared normalat one week and at subsequent time points, and showed no evidence ofinclusion bodies. To further evaluate the mucosa, the epithelium wasbiopsied on day three in the first patient and day one in the secondpatient. Independent evaluation by two pathologists not otherwiseassociated with the study suggested changes consistent with mild traumaand possible ischemia (probably secondary to theanesthetic/vasoconstrictors used before virus administration), but therewere no abnormalities suggestive of virus-mediated damage.

Because the application procedure produced some mild injury in the firsttwo patients, the method of administration was altered in the thirdpatient. The method used did not require the use of local anesthesia orvasoconstriction and which was thus less likely to cause injury, butwhich was also less certain in its ability to constrain Ad2/CFTR-1 in aprecisely defined area. On the right side, Ad2/CFTR-1 was administeredas in the first two patients, and on the left side, the virus wasadministered without anesthesia or the applicator, instead using a smallFoley catheter to apply and maintain Ad2/CFTR-1 in a relatively definedarea by surface tension (FIG. 21E). On the right side, the symptoms andphysical findings were the same as those observed in the first twopatients. By contrast, on the left side there were no symptoms and oninspection the nasal mucosa appeared normal (FIGS. 21D-21F). Nasal swabsobtained from the right side showed neutrophilia similar to thatobserved in the first two patients. In contrast, the left side which hadno anesthesia and minimal manipulation, did not develop neutrophiliaBiopsy of the left side on day 3 after administration (FIG. 22), showedmorphology consistent with CF--a thickened basement membrane andoccasional polymorphonuclear cells in the submucosa--but noabnormalities that could be attributed to the adenovirus vector.

The first patient developed symptoms of a sore throat and increasedcough that began three weeks after treatment and persisted for two days.Six weeks after treatment she developed an exacerbation of herbronchitis/bronchiectasis and hemoptysis that required hospitalization.The second patient had a transient episode of minimal hemoptysis threeweeks after treatment; it was not accompanied by any other symptomsbefore or after the episode. The third patient has an exacerbation ofbronchitis three weeks after treatment for which she was given oralantibiotics. Based on each patient's pretreatment clinical history,evaluation of the episodes, and viral cultures, no evidence could bediscerned that linked these episodes to administration of Ad2/CFTR-1.Rather the episodes appeared consistent with the normal course ofdisease in each individual.

The loss of CFTR Cl⁻ channel function causes abnormal ion transportacross affected epithelia, which in turn contributes to the pathogenesisof CF-associated airway disease (Boat, T. F. et al. in The MetabolicBasis of Inherited Diseases (Scriver, C. R. et al. eds., McGraw-Hill,New York (1989); Quinton, P. M. (1990) FASEBJ 4:2709-2717). In airwayepithelia, ion transport is dominated by two electrically conductiveprocesses: amiloride-sensitive absorption of Na⁺ from the mucosal to thesubmucosal surface and cAMP-stimulated Cl⁻ secretion in the oppositedirection. (Quinton, P. M. (1990) FASEB J 4:2709-2717; Welsh, M. J.(1987) Physio. Rev. 67:1143-1184). These two transport processes can beassessed noninvasively by measuring the voltage across the nasalepithelium (Vt) in vivo (Knowles, M. et al (1981) N. Eng. J. Med.305:1489-1495; Alton, E. W. F. W. et al.(1987) Thorax 42:815-817). FIG.23 shows an example from a normal subject. Under basal conditions, Vtwas electrically negative (lumen referenced to the submucosal surface).Perfusion of amiloride (100 μM) onto the mucosal surface inhibited Vt byblocking apical Na⁺ channels (Knowles, M. et al (1981) N. Eng. J. Med.305:1489-1495; Quinton, P. M. (1990) FAVEB J. 4:2709-2717: Welsh. M. J.(1992) Neuron 8:821-829). Subsequent perfusion of with terbutaline (10μM) a β-adrenergic agonist, hyperpolarized Vt by increasing cellularlevels of cAMP, opening CFTR Cl⁻ channels, and stimulating chloridesecretion (Quinton, P. M. (1990) FASEB J 4:2709-2717; Welsh, M. J. etal. (1992) Neuron 8:821-829). FIG. 24A shows results from seven normalsubjects: basal Vt was -10.5±1.0 mV, and in the presence of amiloride,terbutaline hyperpolarized Vt by -2.3±0.5 mV.

In patients with CF, Vt was more electrically negative than in normalsubjects (FIG. 24B), as has been previously reported (Knowles, M. et al.(1981) N. Eng. J. Med. 305:1489-1495). Basal Vt was -37.0±2.4 mV, muchmore negative than values in normal subjects (P<0.001). (Note thedifference in scale in FIG. 24A and FIG. 24B). Amiloride inhibited Vt,as it did in normal subjects. However, Vt failed to hyperpolarize whenterbutaline was perfused onto the epithelium in the presence ofamiloride. Instead, Vt either did not change or became less negative: onaverage Vt depolarized by +1.8±0.6 mV, a result very different from thatobserved in normal subjects. (P<0.001).

After Ad2/CFTR-1 was applied, basal Vt became less negative in all threeCF patients: FIG. 25A shows an example from the third patient before(FIG. 25A) and after (FIG. 25B) treatment and FIGS. 26A, 26C, and 26Eshow the time course of changes in basal Vt for all three patients. Thedecrease in basal Vt suggests that application of Ad2/CFTR-1 correctedthe CF electolyte transport defect in nasal epithelium of all threepatients. Additional evidence came from an examination of the responseto terbutaline. FIG. 25B shows that in contrast to the response beforeAd2/CFTR-1 was applied, after virus replication, in the presence ofamiloride, terbutaline stimulated Vt. FIGS. 26B, 26D, and 26F show thetime course of the response. These data indicate that Ad2/CFTR-1corrected the CF defect in Cl⁻ transport. Correction of the Cl⁻transport defect cannot be attributed to the anesthesia/applicationprocedure because it did not occur in patients treated with salineinstead of Ad2/CFTR-1 (FIG. 27). Moreover, the effects of the anesthesiawere generalized on the nasal mucosa, but basal Vt decreased only in thearea of virus administration. Finally, similar changes were observed inthe left nasal mucosa of the third patient (FIGS. 26E and 26F), whichhad no symptomatic or physical response after the modified applicationprocedure.

Unsuccessful attempts were made to detect CFTR transcripts by reversetransciptase-PCR and by immunocytochemistry in cells from nasalbrushings and biopsies. Although similar studies in animals have beensuccessful (Zabner, J. et al. (1993) Nature Gen. (in press)), thosestudies used much higher doses of Ad2/CFTR-1. The lack of success in thepresent case likely reflects the small amount of available tissue, thelow MOI, the fact that only a fraction of cells may have been corrected,and the fact that Ad2/CFTR-1 contains a low to moderate strengthpromoter (E1a) which produces much less mRNA and protein than comparableconstructs using a much stronger CMV promoter (unpublished observation).The E1a promoter was chosen because CFTR normally expressed at very lowlevels in airway epithelial cells (Trapnell B. C. et al (1991) Proc.Natl. Acad Sci. USA 88:6565-6569). It is also difficult to detect CFTRprotein and mRNA in normal human airway epithelia, although function isreadily detected because a single ion channel can conduct a very largenumber of ions per second and thus efficiently support Cl⁻ transport.

With time, the electrical changes that indicate correction of the CFdefect reverted toward pretreatment values. However, the basal Vtappeared to revert more slowly than did the change in Vt produced byterbutaline. The significance of this difference is unknown, but it mayreflect the relative sensitivity of the two measurements to expressionof normal CFTR. In any case, this study was not designed to test theduration of correction because the treated area was removed by biopsy onone side and the nasal mucosa on the other side was brushed to obtaincells for analysis at 7 to 10 days after virus administration, and thenat approximately weekly intervals. Brushing the mucosa removes cells,disrupts the epithelium, and reduces basal Vt to zero for at least twodays afterwards, thus preventing an accurate assessment of duration ofthe effect of Ad2/CFTR-1.

Efficacy of adenovirus-mediated gene transfer

The major conclusion of this study is that in vivo application of arecombinant adenovirus encoding CFTR can correct the defect in airwayepithelial Cl⁻ transport that is characteristic of CF epithelia.

Complementation of the Cl⁻ channel defect in human nasal epitheliumcould be measured as a change in basal voltage and as a change in theresponse to cAMP agonists. Although the protocol was not designed toestablish duration, changes in these parameters were detected for atleast three weeks. These results represent the first report thatadministration of a recombinant adenovirus to humans can correct agenetic lesion as measured by a functional assay. This study contrastswith most earlier attempts at gene transfer to humans, in that arecombinant viral vector was administered directly to humans, ratherthan using a in vitro protocol involving removal of cells from thepatient, transduction of the cells in culture, followed byreintroduction of the cells into the patient.

Evidence that the CF Cl⁻ transport defect was corrected at all threedoses of virus, corresponding to 1, 3, and 25 MOI, was obtained. Thisresult is consistent with earlier studies showing that similar MOIsreversed the CF fluid and electrolyte transport defects in primarycultures of CF airway cells grown as epithelia on permeable filtersupports (Rich, D. P. et al. (1993) Human Gene Therapy 4:461-476 andZabner et al. submitted for publication): at an MOI of less than 1,cAMP-stimulated Cl⁻ secretion was partially restored, and aftertreatment with 1 MOI Ad2/CFTR-1 cAMP agonists stimulated fluid secretionthat was within the range observed in epithelia from normal subjects. Atan MOI of 1, a related adenovirus vector produced β-galactosidaseactivity in 20% of infected epithelial cells as assessed byfluorescence-activated cell analysis (Zabner et al. submitted forpublication). Such data would imply that pharmacologic dose ofadenovirus in CF airways might correspond to an MOI of one. If it isestimated that there are 2×10⁶ cells/cm² in the airway (Mariassy, A. T.in Comparative Biology of the Normal Lung (CRC Press, Boca Raton 1992),and that the airways from the trachea to the respiratory bronchioleshave a surface area of 1400 cm² (Weibel, E. R. Morphometry of the HumanLung (Springer Verlag, Heidelberg, 1963) then there would beapproximately 3×10⁹ potential target cells. Assuming a particle to L.U.ratio of 100, this would correspond to approximately 3×10¹¹ particles ofadenovirus with a mass of approximately 75 μg. While obviously only acrude estimate, such information is useful in designing animalexperiments to establish the likely safety profile of a human dose.

It is possible that an efficacious MOI of recombinant adenovirus couldbe less than the lowest MOI tested here. Some evidence suggests that notall cells in an epithelial monolayer need to express CFTR to correct theCF electrolyte transport defects. Mixing experiments showed that whenperhaps 5-10% of cells overexpress CFTR, the monolayer exhibitswild-type electrical properties (Johnson, L. G. et al. (1992) NatureGen. 2:21-25). Studies using liposomes to express CFTR in mice bearing adisrupted CFTR gene also suggest that only a small proportion of cellsneed to be corrected (Hyde, S. C. et al. (1993) Nature 362:250-255). Theresults referred to above using airway epithelial monolayers andmultiplicities of Ad2/CFTR-1 as low as 0.1 showed measurable changes inCl⁻ secretion (Rich, D. P. et al. (1993) Human Gene Therapy 4:461476 andZabner et al. submitted for publication).

Given the very high sensitivity of electrolyte transport assays (whichresult because a single Cl⁻ channel is capable of transporting largenumbers of ions/sec) and the low activity of the E1a promoter used totranscribe CFTR, the inability to detect CFTR protein and CFTR mRNA areperhaps not surprising. Although CFTR mRNA could not be detected byreverse transcriptase-PCR, Ad2/CFTR-1 DNA could be detected in thesamples by standard PCR, demonstrating the presence of input DNA andsuggesting that the reverse transcriptase reaction may have beensuboptimal. This could have occurred because of factors in the tissuethat inhibit the reverse transcriptase. Although there is little doubtthat the changes in electrolyte transport measured here result fromexpression of CFTR, it remains to be seen whether this will lead tomeasurable clinical changes in lung function.

Safety considerations

Application of the adenovirus vector to the nasal epithelium in thesethree patients was well-tolerated. Although mild inflammation wasobserved in the nasal epithelium of all three patients followingadministration of Ad2/CFTR-1, similar changes were observed in twovolunteers who underwent a sham procedure using saline rather than theviral vector. Clearly a combination of anesthetic- and procedure-relatedtrauma resulted in the changes in the nasal mucosa. There isinsufficient evidence to conclude that no inflammation results fromvirus administration. However, using a modified administration of thehighest MOI of virus tested (25 MOI) in one patient, no inflammation wasobserved under conditions that resulted in evidence of biophysicalefficacy that lasted until the area was removed by biopsy at three days.

There was no evidence of replication of Ad2/CFTR-1. Earlier studies hadestablished that replication of Ad2/CFTR-1 in tissue culture andexperimental animals is severely impaired (Rich, D. P. et al. (1993)Human Gene Therapy 4:461476; Zabner, J. et al. (1993) Nature Gen. (inpress)). Replication only occurs in cells that supply the missing earlyproteins of the E1 region of adenovirus, such as 293 cells, or underconditions where the E1 region is provided by coinfection with orrecombination with an E1-containing adenovirus (Graham, F. L. andPrevec, L, Vaccines: New Approaches to Immunological Problems (R. W.Ellis, ed., Boston, Butterworth-Heinermann, 1992); Berkner, K. L. (1988)Biotechniques 6:616-629). The patients studied here where seropositivefor adenovirus types 2 and 5 prior to the study were negative foradenovirus upon culture of nasal swabs prior to administration ofAd2/CFTR-1, and were shown by PCR methods to lack endogenous E1 DNAsequences such as have been reported in some human subjects (Matsuse T.et al. (1992) Am. Rev. Respir. Dis. 146:177-184).

Example 11

Construction and Packaging of Pseudo Adenoviral Vector (PAV)

With reference to FIG. 16, the PAV construct was made by inserting theAd2 packaging signal and E1 enhancer region (0-358 nt) in Bluescript IISK- (Stratagene, LaJolla, Calif.). A variation of this vector, known asPAV II was constructed similarly, except the Ad2 packaging signal and E1enhancer region contained 0-380 nt. The addition of nucleotides at the5' end results in larger PAVs, which may be more efficiently packaged,yet would include more adenoviral sequences and therefore couldpotentially be more immunogenic or more capable of replicating.

To allow ease of manipulation for either the insertion of gene codingregions or complete excision and use in transfections for the purpose ofgenerating infectious particles, a complementary plasmid was also builtin p Bluescript SKII-. This complementary plasmid contains the Ad2 majorlate promoter (MLP) and tripartite leader (TPL) DNA and an SV40T-antigen nuclear localization signal (NLS) and polyadenylation signal(SVpA). As can be seen in FIG. 16, this plasmid contains a convenientrestriction site for the insertion of genes of interest between theMLP/TPL and SV40 poly A. This construct is engineered such that theentire cassette may be excised and inserted into the former PAV I or PAVII construct.

Generation of PAV infectious particles was performed by excision of PAVfrom the plasmid with the Apa I and Sac II restriction endonucleases andco-transfection into 293 cells (an E1a/E1b expressing cell line)(Graham, F. L. et al, (1977) J. Gen Virol 36:59-74) with eitherwild-type Ad2, or packaging/replication deficient helper virus.Purification of PAV from helper can be accompanied by CsCl gradientisolation as PAV viral particles will be of a lower density and willband at a higher position in the gradient.

For gene therapy, it is desirable to generate significant quantities ofPAV virion free from contaminating helper virus. The primary advantageof PAV over standard adenoviral vectors is the ability to package largeDNA inserts into virion (up to about 36 kb). However, PAV requires ahelper virus for replication and packaging and this helper virus will bethe predominant species in any PAV preparation. To increase theproportion of PAV in viral preparation several approaches can beemployed. For example, one can use a helper virus which is partiallydefective for packaging into virions (either by virtue of mutations inthe packaging sequences (Grable, M. and Hearing P. (1992) J. Virol. 66:723-731)) or by virtue of its size -viruses with genome sizes greaterthan approximately 37.5 kb package inefficiently. In mixed infectionswith packaging defective virus, PAV would be expected to be representedat higher levels in the virus mixture than would occur withnon-packaging defective helper viruses.

Another approach is to make the helper virus dependent upon PAV for itsown replication. This may most easily be accomplished by deleting anessential gene from the helper virus (e.g. IX or a terminal protein) andplacing that gene in the PAV vector. In this way neither PAV nor thehelper virus is capable of independent replication--PAV and the helpervirus are therefore co-dependent. This should result in higher PAVrepresentation in the resulting virus preparation.

A third approach is to develop a novel packaging cell line, which iscapable of generating significant quantities of PAV virion free fromcontaminating helper virus. A novel protein IX, (pIX) packaging systemhas been developed. This system exploits several documented features ofadenovirus molecular biology. The first is that adenoviral defectiveparticles are known to comprise up to 30% or more of standard wild-typeadenoviral preparations. These defective or incomplete particles arestable and contain 15-95% of the adenoviral genome, typically 15-30%.Packaging of a PAV genome (15-30% of wild-type genome) should packagecomparably. Secondly, stable packaging of full-length Ad genome but notgenomes <95% required the presence of the adenoviral gene designatedpIX.

The novel packaging system is based on the generation of an Ad proteinpIX expressing 293 cell line. In addition, an adenoviral helper virusengineered such that the E1 region is deleted but enough exogenousmaterial is inserted to equal or slightly exceed the full length 36 kbsize. Both of these two constructs would be introduced into the 293/pIXcell line as purified DNA. In the presence of pIX, yields of bothpredicted progeny viruses as seen in current PAV/Ad2 productionexperiments can be obtained. Virus containing lysates from these cellscan then be titered independently (for the marker gene activity specificto either vector) and used to infect standard 293 (lacking pIX) at amultiplicity of infection of 1 relative to PAV. Since research with thisline as well as from incomplete or defective particle research indicatesthat full length genomes have a competitive packaging advantage, it isexpected that infection with an MOI of 1 relative to PAV willnecessarily equate to an effective MOI for helper of greater than 1. Allcells will presumably contain both PAV (at least 1) and helper (greaterthan 1). Replication and viral capsid production in this cell shouldoccur normally but only PAV genomes should be packaged. Harvesting these93/pIX cultures is expected to yield essentially helper-free PAV.

Example 12

Construction of Ad2-E4/ORF 6

Ad2-E4/ORF6 (FIG. 17 shows the plasmid construction of Ad2-E4/ORF6) isan adenovirus 2 based vector deleted for all Ad2 sequences betweennucleotide 32815 and 35577. This deletion removes all open readingframes of E4 but leaves the E4 promoter and first 32-37 nucleotides ofthe E4 mRNA intact. In place of the deleted sequences, a DNA fragmentencoding ORF6 (Ad2 nucleotides 34082-33178) which was derived bypolymerase chain reaction of Ad2 DNA with ORF6 specific DNA primers(Genzyme oligo. # 2371 -CGGATCCTTTATTATAGGGGAAGTCCACGCCTAC (SEQ. IDNO:8) and oligo. #2372 -CGGGATCCATCGATGAAATATGACTACGTCCG (SEQ. ID NO:9)were inserted). Additional sequences supplied by the oligonucleotidesincluded a cloning site at the 5' and 3' ends of the PCR fragment (Claland BamHl respectively) and a polyadenylation sequence at the 3' end toensure correct polyadenylation of the ORF6 mRNA. As illustrated in FIG.17, the PCR fragment was first ligated to a DNA fragment including theinverted terminal repeat (ITR) and E4 promoter region of Ad2 (Ad2nucleotides 35937-35577) and cloned in the bacterial plasmid pBluescript(Stratagene) to create plasmid ORF6. After sequencing to verify theintegrity of the ORF6 reading frame, the fragment encompassing the ITRand ORF6 was subcloned into a second plasmid, pAd ΔE4, which containsthe 3' end of Ad2 from a Sac I site to the 3' ITR (Ad2 nucleotides28562-35937) and is deleted for all E4 sequences (promoter to poly Asite Ad2 positions 32815-35641) using flunking restriction sites. Inthis second plasmid, virus expressing only E4 ORF6, pAdORF6 was cut withrestriction enzyme PacI and ligated to Ad2 DNA digested with PacI. ThisPacI site corresponds to Ad2 nucleotide 28612. 293 cells weretransfected with the ligation and the resulting virus was subjected torestriction analysis to verify that the Ad2 E4 region had beensubstituted with the corresponding region of pAdORF6 and that the onlyremaining E4 open reading frame was ORF6.

A cell line could in theory be established that would fully complementE4 functions deleted from a recombinant virus. The problem with thisapproach is that E4 functions in the regulation of host cell proteinsynthesis and is therefore toxic to cells. Our current recombinantadenoviruses are deleted for the E1 region and must be grown in 293cells which complement E1 functions. The E4 promoter is activated in bythe E1a gene product, and therefore to prevent inadvertent toxicexpression of E4 transcription of E4 must be tightly regulated. Therequirements of such a promoter or transactivating system is that in theuninduced state expression must be low enough to avoid toxicity to thehost cell, but in the induced state must be sufficiently activated tomake enough E4 gene product to complement the E4 deleted virus duringvirus production.

Example 13

An adenoviral vector is prepared as described in Example 7 whilesubstituting the PGK promoter for the E1a promoter.

Example 14

An adenoviral vector is prepared as described in Example 11 whilesubstituting the PGK promoter for the Ad2 major late promoter (MLP).

Example 15

Generation of Ad2-ORF6/PGK-CFTR

This protocol uses a second generation adenovirus vector namedAd2-ORF6/PGK-CFTR. This virus lacks E1 and in its place contains amodified transcription unit with the phosphoglycerate kinase (PGK)promoter and a poly A addition site flanking the CFTR cDNA. The PGKpromoter is of only moderate strength but is long lasting and notsubject to shut off. The E4 region of the vector has also been modifiedin that the whole coding sequence has been removed and replaced by 0RF6,the only E4 gene essential for growth of Ad in tissue culture. This hasthe effect of generating a genome of 101% the size of wild type Ad2 andrenders the vector more easy to grow in culture than Ad2-ORF6/PGK-CFTR.

The DNA construct comprises a full length copy of the Ad2 genome fromwhich the early region 1 (E1) genes (present at the 5' end of the viralgenome) have been deleted and replaced by an expression cassetteencoding CFTR. The expression cassette includes the promoter forphosphoglycerate kinase (PGK) and a polyadenylation (poly A) additionsignal from the bovine growth hormone gene (BGH). In addition, the E4region of Ad2 has been deleted and replaced with only open reading frame6 (ORF6) of the Ad2 E4 region. The Adenovirus vector is referred to asAD2-ORF6/PGK-CFTR and is illustrated schematically in FIG. 28. Theentire wild-type Ad2 genome has been previously sequenced (Roberts, R.J., (1986) In Adenovirus DNA, W. Oberfler, editor, Matinus NihoffPublishing, Boston) and we have adopted the existing numbering systemwhen referring to the wild type genome. Ad2 genomic regions flanking Eland E4 deletions, and insertions into the genome are being completelysequenced.

The Ad2-ORF6/PGK-CFTR construct differs from the one used in our earlierprotocol (Ad2/CFTR-l) in that the latter utilized the endogenous E1apromoter, had no poly A addition signal directly downstream of CFTR andretained an intact E4 region. The properties of Ad2/CFTR-1 in tissueculture and in animal studies h have been reported (Rich et al., (1993)Human Gene Therapy, 4:461-467; and Zabner et al. (1993) Nature Genetics,In Press).

At the 5' end of the genome, nucleotides 357 to 3328 of Ad2 have beendeleted and replaced with (in order 5' to 3') 22 nucleotides of linker,534 nucleotides of the PGK promoter, 86 nucleotides of linker,nucleotides 123-4622 of the published CFTR sequence (Riordan et al.(1989) Science, 245:1066-1073), 21 nucleotides of linker, and a 32nucleotide synthetic BGH poly A addition signal followed by a final 11nucleotides of linker. The topology of the 5' end of the recombinantmolecule is illustrated in FIG. 28.

At the 3' end of the genome of Ad2-ORF6/PGK-CFTR, Ad2 sequences betweennucleotides 32815 and 35577 have been deleted to remove all open readingframes of E4 but retain the E4 promoter, the E4 cap sites and first32-37 nucleotides of E4 mRNA. The deleted sequences were replaced with afragment derived by PCR which contains open reading frame 6 of Ad2(nucleotides 34082-33178) and a synthetic poly A addition signal. Thetopology of the 3' end of the molecule is shown in FIG. 28. Thepredicted sequence of this region of the molecule is given at the end ofthis appendix. The sequence of this segment of the molecule will beconfirmed. The remainder of the Ad2 viral DNA sequence is published inRoberts, R. J. in Adenovirus DNA. (W. Oberfler, Matinus NihoffPublishing, Boston, 1986 ). The overall size of the Ad2-ORF6/PGK-CFTRvector is 36,336 bp which is 101.3% of full length Ad2.

The CFTR transcript is predicted to initiate at one of three closelyspaced transcriptional start sites in the cloned PGK promoter(Singer-Sam et al. (1984) Gene, 32:409-417) at nucleotides 828, 829 and837 of the recombinant vector (Singer-Sam et al. (1984) Gene,32:409-417). A hybrid 5' untranslated region is comprised of 72, 80 or81 nucleotides of PGK promoter region, 86 nucleotide of linker sequence,and 10 nucleotides derived from the CFTR insert. Transcriptionaltermination is expected to be directed by the BGH poly A addition signalat recombinant vector nucleotide 5530 yielding an approximately 4.7 kbtranscript. The CFTR coding region comprises nucleotides 1010-5454 ofthe recombinant virus and nucleotides 182, 181 or 173 to 4624, 4623, or4615 of the PGK-CFTR-BGH mRNA respectively, depending on whichtranscriptional initiation site is used. Within the CFTR cDNA there aretwo differences from the published (Riordan et al, cited supra) cDNAsequence. An A to C change at position 1990 of the CFTR cDNA (publishedCFTR cDNA coordinates) which was an error in the original publishedsequence, and a T to C change introduced at position 936. The change atposition 936 is translationally silent but increases the stability ofthe cDNA when propagated in bacterial plasmids (Gregory et al. (1990)Nature, 347:382-386; and Cheng et al. (1990) Cell, 63:827-834). The 3'untranslated region of the predicted CFTR transcript comprises 21nucleotides of linker sequence and approximately 10 nucleotides ofsynthetic BGH poly A additional signal.

Although the activity of CFTR can be measured by electrophysiologicalmethods, it is relatively difficult to detect biochemically orimmunocytochemically, particularly at low levels of expression (Gregoryet al., cited supra; and Denning et al. (1992) J. Cell Biol.,118:551-559). A high expression level reporter gene encoding the E. coliβ galactosidase protein fused to a nuclear localization signal derivedfrom the SV40 T-antigen was therefore constructed. Reporter genetranscription is driven by the powerful CMV early gene constitutivepromoter. Specifically, the E1 region of wild type Ad2 betweennucleotides 357-3498 has been deleted and replaced it with a 515 bpfragment containing the CMV promoter and a 3252 bp fragment encoding theβ galactosidase gene.

Regulatory Characteristics of the Elements of the AD2-ORF6/PGK-CFTR

In general terms, the vector is similar to several earlier adenovirusvectors encoding CFTR but it differs in three specific, ways from ourearlier Ad2/CFTR-1 construct.

PGK Promoter

Transcription of CFTR is from the PGK promoter. This is a promoter ofonly moderate strength but because it is a so-called house keepingpromoter we considered it more likely to be capable of long term albeitperhaps low level expression. It may also be less likely to be subjectto "shut-down" than some of the very strong promoters used in otherstudies especially with retroviruses. Since CFTR is not an abundantprotein we believe longevity of expression is probably more criticalthan high level expression. Expression from the PGK promoter in aretrovirus vector has been shown to be long lasting (Apperley et al.(1991) Blood, 78:310-317).

Polyadenylation Signal

Ad2-ORG6/PGK-CFTR contains an exogenous poly A addition signal after theCFTR coding region and prior to the protein IX coding sequence of theAd2 E1 region. Since protein is believed to be involved in packaging ofvirions, we retained this coding region. Furthermore, since protein IXis synthesized from a separate transcript with its own promoter, toprevent possible promoter occlusion at the protein IX promoter, weinserted the BGH poly A addition signal. We have indirect evidence thatpromoter occlusion can be problematic in that Ad2/CMV βGal grows tolower viral titers on 293 cells than does Ad2/βgal-1. These constructsare identical except for the promoter used for β galactosidaseexpression. Since the CMV promoter is much stronger than the E1apromoter we assume that abundant transcription from the CMV promoterthrough the β galactosidase DNA into the protein IX coding regionreduces expression of protein IX from its own promoter by promoterocclusion and that this is responsible for the lower titer ofAd2/CMV-βgal we obtain.

Alterations of the E4 Region

A large portion of the E4 region of the Ad2 genome has been deleted fortwo reasons. The first reason is to decrease the size of the vector usedor expression of CFTR. Adenovirus vectors with genomes much larger thanwild type are packaged less efficiently and are therefore difficult togrow to high titer. The combination of the deletions in the E1 and E4regions in Ad2-ORF6/PGK-CFTR reduce the genome size to 101% of wildtype. In practice we find that it is straightforward to prepare hightier lots of this virus.

The second reason to remove E4 sequences relates to the safety ofadenovirus vectors. It is our goal to remove as many viral genes aspossible to inactive the Ad2 virus backbone in as many ways as possible.The OF 6/7 gene of the E4 region encodes a protein that is involved inactivation of the cellular transcription factor E2-F which is in turnimplicated in the activation of the E2 region of adenovirus (Hemstrom etal. (1991) J. Virol., 65:1440-1449). Therefore removal of ORF6/7 fromadenovirus vectors may provide a further margin of safety at least whengrown in non-proliferating cells. The removal of the El region alreadyrenders such vectors disabled, in part because E1a, if present, is ableto displace E2-F from the retinoblastoma gene product, thereby alsocontributing to the stimulation of E2 transcription. The ORF6 readingframe of Ad2 was added back to the E1-E4 backbone of theAd2-ORF6/PGK-CFTR vector because ORF6 function is essential forproduction of the recombinant virus in 293 cells. ORF6 is believed to beinvolved in DNA replication, host cell shut off and late mRNAaccumulation in the normal adenovirus life cycle. The E1-E4-ORF6⁺backbone Ad2 vector does replicate in 293 cells.

The promoter/enhancer use to drive transcription of ORF6 of E4 is theendogenous E4 promoter. This promoter requires E1a for activation andcontains E1a core enhancer elements and SP1 transcription factor bindingsites (reviewed in Berk, A. J. (1986) Ann. Rev. Genet., 20:75-79).

Replication Origin

The only replication origins present in Ad2-ORF6/PGK-CFTR are thosepresent in the Ad2 parent genome. Replication of Ad2-ORF6/PGK-CFTRsequences has not been detected except when complemented with wild typeE1 activity.

Steps Used to Derive the DNA Construct

Construction of the recombinant Ad2-ORF6/PGK-CFTR virus was accomplishedby in vivo recombination of Ad2-ORF6 DNA and a plasmid containing the 5'10.7 Kb of adenovirus engineered to have an expression cassette encodingthe human CFTR cDNA driven by the PGK promoter and a BGH poly A signalin place of the E1 coding region.

The generation of the plasmid, pBRAd2/PGK/CFTR is described here. Thestarting plasmid contains an approximately 7.5 Kb insert cloned into theClal and BamHI sites of pBR322 and comprises the first 10,680nucleotides of Ad2 with a deletion of the Ad2 sequences betweennucleotides 356 and 3328. This plasmid contains a CMV promoter insertedinto the ClaI and Spel sites at the region of the E1 deletion and isdesignated pBRAd2/CMV. The plasmid also contains the Ad2 5' ITR,packaging and replication sequences and E1 enhancer. The E1 promoter,E1a and most of E1b coding region has been deleted. The 3' terminalportion of the E1b coding region coincides with the pIX promoter whichwas retained. The CMV promoter was removed and replaced with the PGKpromoter as a ClaI and SpeI fragment from the plasmid PGK-GCR. Theresulting plasmid, pBRAd2/PGK, was digested with AvrlI and BstBI and theexcised fragment replaced with the Spel to BstBI fragment from theplasmid construct pAd2E1a/CFTR. This transferred a fragment containingthe CFTR cDNA, BGH poly A signal and the Ad2 genomic sequences from 3327to 10,670. The resulting plasmid is designated pBRAd2/PGK/CFTR. The CFTRcDNA fragment was originally derived from the plasmid pCMV-CFTR-936Cusing restriction enzymes SpeI and Ec113611. pCMV-CFTR-936C consists ofa minimal CFTR cDNA encompassing nucleotides 123-4622 of the publishedCFTR sequence cloned into the multiple cloning site of pRC/CMV(Invitrogen Corp.) using synthetic linkers. The CFTR cDNA within thisplasmid has been completely sequenced.

The Ad2 backbone virus with the E4 region that expresses only openreading frame 6 was constructed as follows. A DNA fragment encoding ORF6(Ad2 nucleotides 34082-33178) was derived by PCR with ORF6 specific DNAprimers. Additional sequences supplied by the oligonucleotides includecloning sites at the 5' and 3' ends of the PCR fragment. (ClaI and BamHIrespectively) and a poly A addition sequence AATAAA at the 3' end toensure correct polyadenylation of ORF6 mRNA. The PCR fragment was clonedinto pBluescript (Stratagene) along with an Ad2 fragment (nucleotides35937-35577) containing the inverted terminal repeat, E4 promoter, E4mRNA cap sites and first 32-37 nucleotides of E4 mRNA to create pORF6. ASalI-BamHI fragment encompassing the ITR and ORF6 was used to replacethe SalI-BamHI fragment encompassing the ITR and E4 deletion in pAdΔE4contains the 3' end of Ad2 from a SpeI site to the 3' ITR (nucleotides27123-35937) and is deleted for all E4 sequences including the promoterand poly A signal (nucleotides 32815-35641). The resulting construct,pAdE40RF6 was cut with PacI and ligated to Ad2 DNA digested with PacInucleotide 28612). 293 cells were transfected with the ligation reactionto generate virus containing only open reading frame 6 from the E4region.

In Vitro Studies with Ad2-ORF6/PGK-CFTR

The ability of Ad2-ORF6/PGK-CFTR to express CFTR in several cell lines,including human HeLa cells, human 293 cells, and primary cultures ofnormal and CF human airway epithelia. As an example, the results fromthe human 293 cells is related here. When human 293 cells were grown onculture dishes, the vector was able to transfer CFTR cDNA and expressCFTR as assessed by immunoprecipitation and by functional assays ofhalide efflux. Gregory, R. J. et al. (1990) Nature 347:382-386; Cheng,S. H. et al. (1990) Cell 63:827-834. More specifically, procedures forpreparing cell lysates, immunoprecipitation of proteins using anti-CFTRantibodies, one-dimensional peptide analysis and SDS-polyacrylamide gelelectrophoresis were as described by Cheng et al. Cheng, S. H. et al.(1990) Cell 63:827-834.

Halide efflux assays were performed as described by Cheng, S. H. et al.(1991) Cell 66:1027-1036. cAMP-stimulated CFTR chloride channel activityusing the halide sensitive fluorophore SPQ in 293 cells treated with 500IU/cell Ad2-ORF6/PGK-CFTR. Stimulation of the infected cells withforskolin (20 μM) and IBMX (100 μm) increased SPQ fluorescenceindicating the presence of functional chloride channels produced by thevector.

Additional studies using primary cultures of human airway (nasal polyp)epithelial cells (from CF patients) infected with Ad2-ORF6/PGK-CFTRdemonstrated that Ad2-ORF6/PGK-CFTR infection of the nasal polypepithelial cells resulted in the expression of cAMP dependent Cl⁻channels. FIG. 29 is an example of the results obtained from suchstudies. Primary cultures of CF nasal polyp epithelial cells wereinfected with Ad2-ORF6/PGK-CFTR at multiplicities of 0.3,3,and 50. Threedays post infection, monlayers were mounted in Ussing chambers andshort-circuit current was measured. At the indicated times: (1) 10 μMamiloride, (2) cAMP agonists (10 μM forskolin and 100 μM IBMX), and (3)1 mM diphenylamine-2-carboxylate were addied to the mucosal solution.

In Vivo Studies with Ad2-ORF6/PGK-CFTR

Virus preparation

Two preparations of Ad2-ORF6/PGK-CFTR virus were used in this study.Both were prepared at Genzyme Corporation, in a Research Laboratory. Thepreparations were purified on a CsCl gradient and then dialyzed againsttris-buffered saline to remove the CsCl. The preparation for the firstadministration (lot #2) had a titer of 2×10¹⁰ IU/ml. The preparation forthe second administration (lot #6) had a titer of 4×10¹⁰ IU/ml.

Animals

Three female Rhesus monkeys, Macaca mulatta, were used for this study.Monkey C (#20046) weighed 6.4 kg. Monkey D (#20047) weighed 6.25 kg.Monkey E (#20048) weighed 10 kg. The monkeys were housed in theUniversity of Iowa at least 360 days before the start of the study. Theanimals were maintained with free access to food and water throughoutthe study. The animals were part of a safety study and efficacy studyfor a different viral vector (Ad2/CFTR-1) and they were exposed to 3nasal viral instillation throughout the year. The previous instillationof Ad2/CFTR-1) has been done 116 days prior to the initiation of thisstudy. All three Rhesus monkeys had an anti-adenoviral antibody responseas detected by ELISA after each viral instillation. There are no knowncontaminants that are expected to interfere with the outcome of thisstudy. Fluorescent lighting was controlled to automatically providealternate light/dark cycles of approximately 12 hours each. The monkeyswere housed in an isolation room in separate cages. Strict respiratoryand body fluid isolation precautions were taken.

Virus administration

For application of the virus, the monkeys were anesthetized byintramuscular injection of ketamine (15 mg/kg). The entire epithelium ofone nasal cavity in each monkey was used for this study. A foleycatheter (size 10) was inserted through each nasal cavity into thepharynx, the balloon was inflated with a 2-3 ml of air, and then pulledanteriorly to obtain a tight occlusion at the posterior choana. TheAd2-ORF6/PGK-CFTR virus was then instilled slowly into the right nostrilwith the posterior balloon inflated. The viral solution remained incontact with the nasal mucosa for 30 min. The balloons were deflated,the catheters were removed, and the monkeys were allowed to recover fromanesthesia.

On the first administration, the viral preparation had a titer of 2×10¹⁰IU/ml. and each monkey received approximately 0.3 ml. Thus the totaldose applied to each monkey was approximately 6.5×10⁹ IU. This totaldose is approximately half the highest dose proposed for the humanstudy. When considered on a IU/kg basis, a 6 kg monkey received a doseapproximately 3 times greater that the highest proposed dose for a 60 kghuman.

Timing of evaluations

The animals were evaluated on the day of administration, and on days 3,7, 24, 38, and 44 days after infection. The second administration ofvirus occurred on day 44. The monkeys were evaluated on day 48 and thenon days 55, 62, and 129.

For evaluations, monkeys were anesthetized by intramuscular injection ofketamine (15 mg/kg). To obtain nasal epithelial cells after the firstviral administration, the nasal mucosa was first impregnated with 5drops of Afrin (0.05% oxymetazoline hydrochloride, Schering-Plough) and1 ml of 2% Lidocaine for 5 minutes. A cytobrush was then used to gentlyrub the mucosa for about 3 sec. To obtain pharyngeal epithelial swabs, acotton-tipped applicator was rubbed over the back of the pharynx 2-3times. The resulting cells were dislodged from brushes or applicatorsinto 2 ml of sterile PBS. After the second administration ofAd2-ORF6/PGK-CFTR, the monkeys were followed clinically for 3 weeks, andmucosal biopsies were obtained from the monkeys medial turbinate at days4, 11 and 18.

Animal evaluation

Animals were evaluated daily for evidence of abnormal behavior ofphysical signs. A record of food and fluid intake was used to assessappetite and general health. Stool consistency was also recorded tocheck for the possibility of diarrhea. At each of the evaluation timepoints, we measured rectal temperature, respiratory rate, and heartrate. The nasal mucosa, conjuctivas and pharynx were visually inspected.The monkeys were also examined for lymphadenopathy.

Hematology and serum chemistry

Venous blood from the monkeys was collected by standard venipuncturetechnique. Blood/serum analysis was performed in the clinical laboratoryof the University of Iowa Hospitals and Clinics using a Hitatchi 737automated chemistry analyzer and a Technicom H6 automated hematologyanalyzer.

Serology

Sera from the monkeys were obtained and antiadenoviral antibody titerswere measured by ELISA. For the ELISA, 50 ng/well of killed adenovirus(Lee Biomolecular Research Laboratories, San Diego, Calif.) was coatedin 0.1M NaHCO₃ at 4° C. overnight on 96 well plates. The test samples atappropriate dillutions were added, starting at a dillution of 1/50. Thesamples were incubated for 1 hour, the plates washed, and a Goatanti-human IgG HRP conjugate (Jackson ImmunoResearch Laboratories, WestGrove, Pa.) was added for 1 hour. The plates were washed andO-Phenylenediamine (OPD) (Sigma Chemical Co., St. Louis, Mo.) was addedfor 30 min. at room temperature. The assay was stopped with 4.5M H₂ SO₄and read at 490 nm on a Molecular Devises microplate reader. The titerwas calculated as the product of the reciprocal of the initial dilutionand the reciprocal of the dilution in the last well with an OD>0.100.Nasal washings from the monkeys were obtained and antiadenoviralantibody titers were measured by ELISA, starting at a dilution of 1/4.

Nasal Washings

Nasal washings were obtained to test for the possibility of secretoryantibodies that could act as neutralizing antibodies. Three ml ofsterile PBS as slowly instilled into the nasal cavity of the monkeys,the fluid was collected by gravity. The washings were centrifuged at1000 RPM for 5 minutes and the supernatant was used for anti-adenoviral,and neutralizing antibody measurement.

Cytolog

Cells were obtained from the monkey's nasal epithelium by gently rubbingthe nasal mucosa for about 3 seconds with a cytobrush. The resultingcells were dislodged from the brushes into 2 ml of PBS. The cellsuspension was spun at 5000 rpm for 5 min. and resuspended in 293 mediaat a concentration of 10⁶ cells/ml. Forty μl of the cell suspension wasplaced on slides using a Cytospin. Cytospin slides were stained withWright's stain and analyzed for cell differential using lightmicroscopy.

Culture for Ad2-ORF6/PFK-CFTRB

To assess for the presence of infectious viral particles, thesupernatant from the nasal brushings and pharyngeal swabs of the monkeyswere used. Twenty-five μL of the supernatant was added in duplicate to293 cells. 293 cells were used at 50% confluence and were seeded in 96well plates. 293 cells were incubated for 72 hours at 37° C., then fixedwith a mixture of equal parts of methanol and acetone for 10 min andincubated with an FITC label antiadenovirus monoclonal antibodies(Chemicon, Light Diagnostics, Temecuca, Calif.) for 30 min. Positivenuclear immunofluorescence was interpreted as positive culture.

Immunocytochemistry for the detection of CFTR

Cells were obtained by brushing. Eighty μI of cell suspension were spunonto gelatin-coated slides. The slides were allowed to air dry, and thenfixed with 4% paraformaldehyde. The cells were permeabilized with 0.2Triton-X (Pierce, Rockford, Ill.) and then blocked for 60 minutes with5% goat serum (Sigma, Mo.). A pool of monoclonal antibodies (M13-1,M1-4, and M6-4) (Gregory et al., 1990; Denning et al., 1992b; Denning etal., 1992a) were added and incubated for 12 hours. The primary antibodywas washed off and an antimouse biotinylated antibody (Biomeda, FosterCity, Calif.) was added. After washing, the secondary antibody,streptavidin FITC (Biomeda, Foster City, Calif.) was added and theslides were observed with a laser scanning confocal microscope.

Biopsies

To assess for histologic evidence of safety, nasal medial turbinatebiopsies were obtained on day 4, 11 and 18 after the second viraladministration as described before (Zabner et al (1993) Human GeneTherapy, in press). Nasal biopsies were fixed in 4% formaldehyde and H&Estained sections were reviewed.

RESULTS

Studies of efficacy

To directly assess the presence of CFTR, cells obtained by brushing wereplated onto slides by cytospin and stained with antibodies to CFTR. Apositive reaction is clearly evident in cells exposed toAd2-ORF6/PGK-CFTR. The cells were scored as positive byimmunocytochemistry when evaluated by a reader blinded to the identityof the samples. Cells obtained prior to infection and from otheruntreated monkeys were used as negative controls.

Studies of safety

None of the monkeys developed any clinical signs of viral infections orinflammation. There were no visible abnormalities at days 3, 4, 7 or onweekly inspection thereafter. Physical examination revealed no fever,lymphadenopathy, conjunctivitis, ocryza, tachypnea, or tachycardia atany of the time points. There was no cough, sneezing or diarrhea. Themonkeys had no fever. Appetites and weights were not affected by virusadministration in either monkey. The data are summarized in FIGS.30A-30C.

The presence of live virus was tested in the supernatant of cellsuspensions from swabs and brushes from each nostril and the pharynx.Each supernatant was used to infect the virus-sensitive 293 cell line.Live virus was never detected at any of the time points. The rapid lossof live virus suggests that there was no viral replication.

The results of complete blood counts, sedimentation rate, and clinicalchemistries are shown in FIG. 31A-31C. There was no evidence of asystemic inflammatory response or other abnormalities of the clinicalchemistries.

Epithelial inflammation was assessed by cytological examination ofWright-stained cells (cytospin) obtained from brushings of the nasalepithelium. The percentage of neutrophils and lymphocytes from theinfected nostrils were compared to those of the control nostrils andvalues from four control monkeys. Wright stains of cells from nasalbrushing were performed on each of the evaluation days. Neutrophils andlymphocytes accounted for less than 5% of total cells at all timepoints. The data are shown in FIGS. 32A-32C. The data indicate thatadministration of Ad2-ORF6/PGK-CFTR caused no change in the distributionor number of inflammatory cells at any of the time points followingvirus administration, even during a second administration of the virus.The biopsies slides obtained after the second Ad2-ORF6/PGK-CFTRadministration were reviewed by an independent pathologist, who found noevidence of inflammation or any other cytopathic effects.

FIGS. 33A-33C shows that all three monkeys had developed antibody titersto adenovirus prior to the first infection with Ad2-ORF6/PGK-CFTR(Zabner et al. (1993) Human Gene Therapy (in press)). Antibody titersmeasured by ELISA rose within one week after the first and secondadministration and peaked at day 24. No antiadenoviral antibodies weredetected by ELISA or neutralizing assay in nasal washings of any of themonkeys.

These results combined with demonstrate the ability of a recombinantadenovirus encoding CFTR (Ad2-ORF6/PGK-CFTR) to express CFTR cDNA in theairway epithelium of monkeys. These monkeys have been followedclinically for 12 months after the first viral administration and nocomplications have been observed.

The results of the safety studies are encouraging. We found no evidenceof viral replication; infectious viral particles were rapidly cleared.The other major consideration for safety of an adenovirus vector in thetreatment of CF is the possibility of an inflammatory response. The dataindicate that the virus generated an antibody response, but despitethis, we observed no evidence of a systemic or local inflammatoryresponse. The cells obtained by brushings and swabs were not altered byvirus application. Since these Monkeys had been previously exposed threetimes to Ad2/CFTR- 1, these data suggests that at least five sequentialexposures of airway epithelium to adenovirus does not cause adetrimental inflammatory response.

These data indicate that Ad2-ORF6/PGK-CFTR can effectively transfer CFTRcDNA to airway epithelium and direct the expression of CFTR. They alsoindicate that transfer and expression is safe in primates.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 10    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6129 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 133..4572    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCA60    GAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGC120    GCCCGAGAGACCATGCAGAGGTCGCCTCTGGAAAAGGCCAGCGTTGTC168    MetGlnArgSerProLeuGluLysAlaSerValVal    1510    TCCAAACTTTTTTTCAGCTGGACCAGACCAATTTTGAGGAAAGGATAC216    SerLysLeuPhePheSerTrpThrArgProIleLeuArgLysGlyTyr    152025    AGACAGCGCCTGGAATTGTCAGACATATACCAAATCCCTTCTGTTGAT264    ArgGlnArgLeuGluLeuSerAspIleTyrGlnIleProSerValAsp    303540    TCTGCTGACAATCTATCTGAAAAATTGGAAAGAGAATGGGATAGAGAG312    SerAlaAspAsnLeuSerGluLysLeuGluArgGluTrpAspArgGlu    45505560    CTGGCTTCAAAGAAAAATCCTAAACTCATTAATGCCCTTCGGCGATGT360    LeuAlaSerLysLysAsnProLysLeuIleAsnAlaLeuArgArgCys    657075    TTTTTCTGGAGATTTATGTTCTATGGAATCTTTTTATATTTAGGGGAA408    PhePheTrpArgPheMetPheTyrGlyIlePheLeuTyrLeuGlyGlu    808590    GTCACCAAAGCAGTACAGCCTCTCTTACTGGGAAGAATCATAGCTTCC456    ValThrLysAlaValGlnProLeuLeuLeuGlyArgIleIleAlaSer    95100105    TATGACCCGGATAACAAGGAGGAACGCTCTATCGCGATTTATCTAGGC504    TyrAspProAspAsnLysGluGluArgSerIleAlaIleTyrLeuGly    110115120    ATAGGCTTATGCCTTCTCTTTATTGTGAGGACACTGCTCCTACACCCA552    IleGlyLeuCysLeuLeuPheIleValArgThrLeuLeuLeuHisPro    125130135140    GCCATTTTTGGCCTTCATCACATTGGAATGCAGATGAGAATAGCTATG600    AlaIlePheGlyLeuHisHisIleGlyMetGlnMetArgIleAlaMet    145150155    TTTAGTTTGATTTATAAGAAGACTTTAAAGCTGTCAAGCCGTGTTCTA648    PheSerLeuIleTyrLysLysThrLeuLysLeuSerSerArgValLeu    160165170    GATAAAATAAGTATTGGACAACTTGTTAGTCTCCTTTCCAACAACCTG696    AspLysIleSerIleGlyGlnLeuValSerLeuLeuSerAsnAsnLeu    175180185    AACAAATTTGATGAAGGACTTGCATTGGCACATTTCGTGTGGATCGCT744    AsnLysPheAspGluGlyLeuAlaLeuAlaHisPheValTrpIleAla    190195200    CCTTTGCAAGTGGCACTCCTCATGGGGCTAATCTGGGAGTTGTTACAG792    ProLeuGlnValAlaLeuLeuMetGlyLeuIleTrpGluLeuLeuGln    205210215220    GCGTCTGCCTTCTGTGGACTTGGTTTCCTGATAGTCCTTGCCCTTTTT840    AlaSerAlaPheCysGlyLeuGlyPheLeuIleValLeuAlaLeuPhe    225230235    CAGGCTGGGCTAGGGAGAATGATGATGAAGTACAGAGATCAGAGAGCT888    GlnAlaGlyLeuGlyArgMetMetMetLysTyrArgAspGlnArgAla    240245250    GGGAAGATCAGTGAAAGACTTGTGATTACCTCAGAAATGATTGAAAAT936    GlyLysIleSerGluArgLeuValIleThrSerGluMetIleGluAsn    255260265    ATCCAATCTGTTAAGGCATACTGCTGGGAAGAAGCAATGGAAAAAATG984    IleGlnSerValLysAlaTyrCysTrpGluGluAlaMetGluLysMet    270275280    ATTGAAAACTTAAGACAAACAGAACTGAAACTGACTCGGAAGGCAGCC1032    IleGluAsnLeuArgGlnThrGluLeuLysLeuThrArgLysAlaAla    285290295300    TATGTGAGATACTTCAATAGCTCAGCCTTCTTCTTCTCAGGGTTCTTT1080    TyrValArgTyrPheAsnSerSerAlaPhePhePheSerGlyPhePhe    305310315    GTGGTGTTTTTATCTGTGCTTCCCTATGCACTAATCAAAGGAATCATC1128    ValValPheLeuSerValLeuProTyrAlaLeuIleLysGlyIleIle    320325330    CTCCGGAAAATATTCACCACCATCTCATTCTGCATTGTTCTGCGCATG1176    LeuArgLysIlePheThrThrIleSerPheCysIleValLeuArgMet    335340345    GCGGTCACTCGGCAATTTCCCTGGGCTGTACAAACATGGTATGACTCT1224    AlaValThrArgGlnPheProTrpAlaValGlnThrTrpTyrAspSer    350355360    CTTGGAGCAATAAACAAAATACAGGATTTCTTACAAAAGCAAGAATAT1272    LeuGlyAlaIleAsnLysIleGlnAspPheLeuGlnLysGlnGluTyr    365370375380    AAGACATTGGAATATAACTTAACGACTACAGAAGTAGTGATGGAGAAT1320    LysThrLeuGluTyrAsnLeuThrThrThrGluValValMetGluAsn    385390395    GTAACAGCCTTCTGGGAGGAGGGATTTGGGGAATTATTTGAGAAAGCA1368    ValThrAlaPheTrpGluGluGlyPheGlyGluLeuPheGluLysAla    400405410    AAACAAAACAATAACAATAGAAAAACTTCTAATGGTGATGACAGCCTC1416    LysGlnAsnAsnAsnAsnArgLysThrSerAsnGlyAspAspSerLeu    415420425    TTCTTCAGTAATTTCTCACTTCTTGGTACTCCTGTCCTGAAAGATATT1464    PhePheSerAsnPheSerLeuLeuGlyThrProValLeuLysAspIle    430435440    AATTTCAAGATAGAAAGAGGACAGTTGTTGGCGGTTGCTGGATCCACT1512    AsnPheLysIleGluArgGlyGlnLeuLeuAlaValAlaGlySerThr    445450455460    GGAGCAGGCAAGACTTCACTTCTAATGATGATTATGGGAGAACTGGAG1560    GlyAlaGlyLysThrSerLeuLeuMetMetIleMetGlyGluLeuGlu    465470475    CCTTCAGAGGGTAAAATTAAGCACAGTGGAAGAATTTCATTCTGTTCT1608    ProSerGluGlyLysIleLysHisSerGlyArgIleSerPheCysSer    480485490    CAGTTTTCCTGGATTATGCCTGGCACCATTAAAGAAAATATCATCTTT1656    GlnPheSerTrpIleMetProGlyThrIleLysGluAsnIleIlePhe    495500505    GGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAAAGCATGC1704    GlyValSerTyrAspGluTyrArgTyrArgSerValIleLysAlaCys    510515520    CAACTAGAAGAGGACATCTCCAAGTTTGCAGAGAAAGACAATATAGTT1752    GlnLeuGluGluAspIleSerLysPheAlaGluLysAspAsnIleVal    525530535540    CTTGGAGAAGGTGGAATCACACTGAGTGGAGGTCAACGAGCAAGAATT1800    LeuGlyGluGlyGlyIleThrLeuSerGlyGlyGlnArgAlaArgIle    545550555    TCTTTAGCAAGAGCAGTATACAAAGATGCTGATTTGTATTTATTAGAC1848    SerLeuAlaArgAlaValTyrLysAspAlaAspLeuTyrLeuLeuAsp    560565570    TCTCCTTTTGGATACCTAGATGTTTTAACAGAAAAAGAAATATTTGAA1896    SerProPheGlyTyrLeuAspValLeuThrGluLysGluIlePheGlu    575580585    AGCTGTGTCTGTAAACTGATGGCTAACAAAACTAGGATTTTGGTCACT1944    SerCysValCysLysLeuMetAlaAsnLysThrArgIleLeuValThr    590595600    TCTAAAATGGAACATTTAAAGAAAGCTGACAAAATATTAATTTTGCAT1992    SerLysMetGluHisLeuLysLysAlaAspLysIleLeuIleLeuHis    605610615620    GAAGGTAGCAGCTATTTTTATGGGACATTTTCAGAACTCCAAAATCTA2040    GluGlySerSerTyrPheTyrGlyThrPheSerGluLeuGlnAsnLeu    625630635    CAGCCAGACTTTAGCTCAAAACTCATGGGATGTGATTCTTTCGACCAA2088    GlnProAspPheSerSerLysLeuMetGlyCysAspSerPheAspGln    640645650    TTTAGTGCAGAAAGAAGAAATTCAATCCTAACTGAGACCTTACACCGT2136    PheSerAlaGluArgArgAsnSerIleLeuThrGluThrLeuHisArg    655660665    TTCTCATTAGAAGGAGATGCTCCTGTCTCCTGGACAGAAACAAAAAAA2184    PheSerLeuGluGlyAspAlaProValSerTrpThrGluThrLysLys    670675680    CAATCTTTTAAACAGACTGGAGAGTTTGGGGAAAAAAGGAAGAATTCT2232    GlnSerPheLysGlnThrGlyGluPheGlyGluLysArgLysAsnSer    685690695700    ATTCTCAATCCAATCAACTCTATACGAAAATTTTCCATTGTGCAAAAG2280    IleLeuAsnProIleAsnSerIleArgLysPheSerIleValGlnLys    705710715    ACTCCCTTACAAATGAATGGCATCGAAGAGGATTCTGATGAGCCTTTA2328    ThrProLeuGlnMetAsnGlyIleGluGluAspSerAspGluProLeu    720725730    GAGAGAAGGCTGTCCTTAGTACCAGATTCTGAGCAGGGAGAGGCGATA2376    GluArgArgLeuSerLeuValProAspSerGluGlnGlyGluAlaIle    735740745    CTGCCTCGCATCAGCGTGATCAGCACTGGCCCCACGCTTCAGGCACGA2424    LeuProArgIleSerValIleSerThrGlyProThrLeuGlnAlaArg    750755760    AGGAGGCAGTCTGTCCTGAACCTGATGACACACTCAGTTAACCAAGGT2472    ArgArgGlnSerValLeuAsnLeuMetThrHisSerValAsnGlnGly    765770775780    CAGAACATTCACCGAAAGACAACAGCATCCACACGAAAAGTGTCACTG2520    GlnAsnIleHisArgLysThrThrAlaSerThrArgLysValSerLeu    785790795    GCCCCTCAGGCAAACTTGACTGAACTGGATATATATTCAAGAAGGTTA2568    AlaProGlnAlaAsnLeuThrGluLeuAspIleTyrSerArgArgLeu    800805810    TCTCAAGAAACTGGCTTGGAAATAAGTGAAGAAATTAACGAAGAAGAC2616    SerGlnGluThrGlyLeuGluIleSerGluGluIleAsnGluGluAsp    815820825    TTAAAGGAGTGCCTTTTTGATGATATGGAGAGCATACCAGCAGTGACT2664    LeuLysGluCysLeuPheAspAspMetGluSerIleProAlaValThr    830835840    ACATGGAACACATACCTTCGATATATTACTGTCCACAAGAGCTTAATT2712    ThrTrpAsnThrTyrLeuArgTyrIleThrValHisLysSerLeuIle    845850855860    TTTGTGCTAATTTGGTGCTTAGTAATTTTTCTGGCAGAGGTGGCTGCT2760    PheValLeuIleTrpCysLeuValIlePheLeuAlaGluValAlaAla    865870875    TCTTTGGTTGTGCTGTGGCTCCTTGGAAACACTCCTCTTCAAGACAAA2808    SerLeuValValLeuTrpLeuLeuGlyAsnThrProLeuGlnAspLys    880885890    GGGAATAGTACTCATAGTAGAAATAACAGCTATGCAGTGATTATCACC2856    GlyAsnSerThrHisSerArgAsnAsnSerTyrAlaValIleIleThr    895900905    AGCACCAGTTCGTATTATGTGTTTTACATTTACGTGGGAGTAGCCGAC2904    SerThrSerSerTyrTyrValPheTyrIleTyrValGlyValAlaAsp    910915920    ACTTTGCTTGCTATGGGATTCTTCAGAGGTCTACCACTGGTGCATACT2952    ThrLeuLeuAlaMetGlyPhePheArgGlyLeuProLeuValHisThr    925930935940    CTAATCACAGTGTCGAAAATTTTACACCACAAAATGTTACATTCTGTT3000    LeuIleThrValSerLysIleLeuHisHisLysMetLeuHisSerVal    945950955    CTTCAAGCACCTATGTCAACCCTCAACACGTTGAAAGCAGGTGGGATT3048    LeuGlnAlaProMetSerThrLeuAsnThrLeuLysAlaGlyGlyIle    960965970    CTTAATAGATTCTCCAAAGATATAGCAATTTTGGATGACCTTCTGCCT3096    LeuAsnArgPheSerLysAspIleAlaIleLeuAspAspLeuLeuPro    975980985    CTTACCATATTTGACTTCATCCAGTTGTTATTAATTGTGATTGGAGCT3144    LeuThrIlePheAspPheIleGlnLeuLeuLeuIleValIleGlyAla    9909951000    ATAGCAGTTGTCGCAGTTTTACAACCCTACATCTTTGTTGCAACAGTG3192    IleAlaValValAlaValLeuGlnProTyrIlePheValAlaThrVal    1005101010151020    CCAGTGATAGTGGCTTTTATTATGTTGAGAGCATATTTCCTCCAAACC3240    ProValIleValAlaPheIleMetLeuArgAlaTyrPheLeuGlnThr    102510301035    TCACAGCAACTCAAACAACTGGAATCTGAAGGCAGGAGTCCAATTTTC3288    SerGlnGlnLeuLysGlnLeuGluSerGluGlyArgSerProIlePhe    104010451050    ACTCATCTTGTTACAAGCTTAAAAGGACTATGGACACTTCGTGCCTTC3336    ThrHisLeuValThrSerLeuLysGlyLeuTrpThrLeuArgAlaPhe    105510601065    GGACGGCAGCCTTACTTTGAAACTCTGTTCCACAAAGCTCTGAATTTA3384    GlyArgGlnProTyrPheGluThrLeuPheHisLysAlaLeuAsnLeu    107010751080    CATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCCAA3432    HisThrAlaAsnTrpPheLeuTyrLeuSerThrLeuArgTrpPheGln    1085109010951100    ATGAGAATAGAAATGATTTTTGTCATCTTCTTCATTGCTGTTACCTTC3480    MetArgIleGluMetIlePheValIlePhePheIleAlaValThrPhe    110511101115    ATTTCCATTTTAACAACAGGAGAAGGAGAAGGAAGAGTTGGTATTATC3528    IleSerIleLeuThrThrGlyGluGlyGluGlyArgValGlyIleIle    112011251130    CTGACTTTAGCCATGAATATCATGAGTACATTGCAGTGGGCTGTAAAC3576    LeuThrLeuAlaMetAsnIleMetSerThrLeuGlnTrpAlaValAsn    113511401145    TCCAGCATAGATGTGGATAGCTTGATGCGATCTGTGAGCCGAGTCTTT3624    SerSerIleAspValAspSerLeuMetArgSerValSerArgValPhe    115011551160    AAGTTCATTGACATGCCAACAGAAGGTAAACCTACCAAGTCAACCAAA3672    LysPheIleAspMetProThrGluGlyLysProThrLysSerThrLys    1165117011751180    CCATACAAGAATGGCCAACTCTCGAAAGTTATGATTATTGAGAATTCA3720    ProTyrLysAsnGlyGlnLeuSerLysValMetIleIleGluAsnSer    118511901195    CACGTGAAGAAAGATGACATCTGGCCCTCAGGGGGCCAAATGACTGTC3768    HisValLysLysAspAspIleTrpProSerGlyGlyGlnMetThrVal    120012051210    AAAGATCTCACAGCAAAATACACAGAAGGTGGAAATGCCATATTAGAG3816    LysAspLeuThrAlaLysTyrThrGluGlyGlyAsnAlaIleLeuGlu    121512201225    AACATTTCCTTCTCAATAAGTCCTGGCCAGAGGGTGGGCCTCTTGGGA3864    AsnIleSerPheSerIleSerProGlyGlnArgValGlyLeuLeuGly    123012351240    AGAACTGGATCAGGGAAGAGTACTTTGTTATCAGCTTTTTTGAGACTA3912    ArgThrGlySerGlyLysSerThrLeuLeuSerAlaPheLeuArgLeu    1245125012551260    CTGAACACTGAAGGAGAAATCCAGATCGATGGTGTGTCTTGGGATTCA3960    LeuAsnThrGluGlyGluIleGlnIleAspGlyValSerTrpAspSer    126512701275    ATAACTTTGCAACAGTGGAGGAAAGCCTTTGGAGTGATACCACAGAAA4008    IleThrLeuGlnGlnTrpArgLysAlaPheGlyValIleProGlnLys    128012851290    GTATTTATTTTTTCTGGAACATTTAGAAAAAACTTGGATCCCTATGAA4056    ValPheIlePheSerGlyThrPheArgLysAsnLeuAspProTyrGlu    129513001305    CAGTGGAGTGATCAAGAAATATGGAAAGTTGCAGATGAGGTTGGGCTC4104    GlnTrpSerAspGlnGluIleTrpLysValAlaAspGluValGlyLeu    131013151320    AGATCTGTGATAGAACAGTTTCCTGGGAAGCTTGACTTTGTCCTTGTG4152    ArgSerValIleGluGlnPheProGlyLysLeuAspPheValLeuVal    1325133013351340    GATGGGGGCTGTGTCCTAAGCCATGGCCACAAGCAGTTGATGTGCTTG4200    AspGlyGlyCysValLeuSerHisGlyHisLysGlnLeuMetCysLeu    134513501355    GCTAGATCTGTTCTCAGTAAGGCGAAGATCTTGCTGCTTGATGAACCC4248    AlaArgSerValLeuSerLysAlaLysIleLeuLeuLeuAspGluPro    136013651370    AGTGCTCATTTGGATCCAGTAACATACCAAATAATTAGAAGAACTCTA4296    SerAlaHisLeuAspProValThrTyrGlnIleIleArgArgThrLeu    137513801385    AAACAAGCATTTGCTGATTGCACAGTAATTCTCTGTGAACACAGGATA4344    LysGlnAlaPheAlaAspCysThrValIleLeuCysGluHisArgIle    139013951400    GAAGCAATGCTGGAATGCCAACAATTTTTGGTCATAGAAGAGAACAAA4392    GluAlaMetLeuGluCysGlnGlnPheLeuValIleGluGluAsnLys    1405141014151420    GTGCGGCAGTACGATTCCATCCAGAAACTGCTGAACGAGAGGAGCCTC4440    ValArgGlnTyrAspSerIleGlnLysLeuLeuAsnGluArgSerLeu    142514301435    TTCCGGCAAGCCATCAGCCCCTCCGACAGGGTGAAGCTCTTTCCCCAC4488    PheArgGlnAlaIleSerProSerAspArgValLysLeuPheProHis    144014451450    CGGAACTCAAGCAAGTGCAAGTCTAAGCCCCAGATTGCTGCTCTGAAA4536    ArgAsnSerSerLysCysLysSerLysProGlnIleAlaAlaLeuLys    145514601465    GAGGAGACAGAAGAAGAGGTGCAAGATACAAGGCTTTAGAGAGCAG4582    GluGluThrGluGluGluValGlnAspThrArgLeu    147014751480    CATAAATGTTGACATGGGACATTTGCTCATGGAATTGGAGCTCGTGGGACAGTCACCTCA4642    TGGAATTGGAGCTCGTGGAACAGTTACCTCTGCCTCAGAAAACAAGGATGAATTAAGTTT4702    TTTTTTAAAAAAGAAACATTTGGTAAGGGGAATTGAGGACACTGATATGGGTCTTGATAA4762    ATGGCTTCCTGGCAATAGTCAAATTGTGTGAAAGGTACTTCAAATCCTTGAAGATTTACC4822    ACTTGTGTTTTGCAAGCCAGATTTTCCTGAAAACCCTTGCCATGTGCTAGTAATTGGAAA4882    GGCAGCTCTAAATGTCAATCAGCCTAGTTGATCAGCTTATTGTCTAGTGAAACTCGTTAA4942    TTTGTAGTGTTGGAGAAGAACTGAAATCATACTTCTTAGGGTTATGATTAAGTAATGATA5002    ACTGGAAACTTCAGCGGTTTATATAAGCTTGTATTCCTTTTTCTCTCCTCTCCCCATGAT5062    GTTTAGAAACACAACTATATTGTTTGCTAAGCATTCCAACTATCTCATTTCCAAGCAAGT5122    ATTAGAATACCACAGGAACCACAAGACTGCACATCAAAATATGCCCCATTCAACATCTAG5182    TGAGCAGTCAGGAAAGAGAACTTCCAGATCCTGGAAATCAGGGTTAGTATTGTCCAGGTC5242    TACCAAAAATCTCAATATTTCAGATAATCACAATACATCCCTTACCTGGGAAAGGGCTGT5302    TATAATCTTTCACAGGGGACAGGATGGTTCCCTTGATGAAGAAGTTGATATGCCTTTTCC5362    CAACTCCAGAAAGTGACAAGCTCACAGACCTTTGAACTAGAGTTTAGCTGGAAAAGTATG5422    TTAGTGCAAATTGTCACAGGACAGCCCTTCTTTCCACAGAAGCTCCAGGTAGAGGGTGTG5482    TAAGTAGATAGGCCATGGGCACTGTGGGTAGACACACATGAAGTCCAAGCATTTAGATGT5542    ATAGGTTGATGGTGGTATGTTTTCAGGCTAGATGTATGTACTTCATGCTGTCTACACTAA5602    GAGAGAATGAGAGACACACTGAAGAAGCACCAATCATGAATTAGTTTTATATGCTTCTGT5662    TTTATAATTTTGTGAAGCAAAATTTTTTCTCTAGGAAATATTTATTTTAATAATGTTTCA5722    AACATATATTACAATGCTGTATTTTAAAAGAATGATTATGAATTACATTTGTATAAAATA5782    ATTTTTATATTTGAAATATTGACTTTTTATGGCACTAGTATTTTTATGAAATATTATGTT5842    AAAACTGGGACAGGGGAGAACCTAGGGTGATATTAACCAGGGGCCATGAATCACCTTTTG5902    GTCTGGAGGGAAGCCTTGGGGCTGATCGAGTTGTTGCCCACAGCTGTATGATTCCCAGCC5962    AGACACAGCCTCTTAGATGCAGTTCTGAAGAAGATGGTACCACCAGTCTGACTGTTTCCA6022    TCAAGGGTACACTGCCTTCTCAACTCCAAACTGACTCTTAAGAAGACTGCATTATATTTA6082    TTACTGTAAGAAAATATCACTTGTCAATAAAATCCATACATTTGTGT6129    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1480 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetGlnArgSerProLeuGluLysAlaSerValValSerLysLeuPhe    151015    PheSerTrpThrArgProIleLeuArgLysGlyTyrArgGlnArgLeu    202530    GluLeuSerAspIleTyrGlnIleProSerValAspSerAlaAspAsn    354045    LeuSerGluLysLeuGluArgGluTrpAspArgGluLeuAlaSerLys    505560    LysAsnProLysLeuIleAsnAlaLeuArgArgCysPhePheTrpArg    65707580    PheMetPheTyrGlyIlePheLeuTyrLeuGlyGluValThrLysAla    859095    ValGlnProLeuLeuLeuGlyArgIleIleAlaSerTyrAspProAsp    100105110    AsnLysGluGluArgSerIleAlaIleTyrLeuGlyIleGlyLeuCys    115120125    LeuLeuPheIleValArgThrLeuLeuLeuHisProAlaIlePheGly    130135140    LeuHisHisIleGlyMetGlnMetArgIleAlaMetPheSerLeuIle    145150155160    TyrLysLysThrLeuLysLeuSerSerArgValLeuAspLysIleSer    165170175    IleGlyGlnLeuValSerLeuLeuSerAsnAsnLeuAsnLysPheAsp    180185190    GluGlyLeuAlaLeuAlaHisPheValTrpIleAlaProLeuGlnVal    195200205    AlaLeuLeuMetGlyLeuIleTrpGluLeuLeuGlnAlaSerAlaPhe    210215220    CysGlyLeuGlyPheLeuIleValLeuAlaLeuPheGlnAlaGlyLeu    225230235240    GlyArgMetMetMetLysTyrArgAspGlnArgAlaGlyLysIleSer    245250255    GluArgLeuValIleThrSerGluMetIleGluAsnIleGlnSerVal    260265270    LysAlaTyrCysTrpGluGluAlaMetGluLysMetIleGluAsnLeu    275280285    ArgGlnThrGluLeuLysLeuThrArgLysAlaAlaTyrValArgTyr    290295300    PheAsnSerSerAlaPhePhePheSerGlyPhePheValValPheLeu    305310315320    SerValLeuProTyrAlaLeuIleLysGlyIleIleLeuArgLysIle    325330335    PheThrThrIleSerPheCysIleValLeuArgMetAlaValThrArg    340345350    GlnPheProTrpAlaValGlnThrTrpTyrAspSerLeuGlyAlaIle    355360365    AsnLysIleGlnAspPheLeuGlnLysGlnGluTyrLysThrLeuGlu    370375380    TyrAsnLeuThrThrThrGluValValMetGluAsnValThrAlaPhe    385390395400    TrpGluGluGlyPheGlyGluLeuPheGluLysAlaLysGlnAsnAsn    405410415    AsnAsnArgLysThrSerAsnGlyAspAspSerLeuPhePheSerAsn    420425430    PheSerLeuLeuGlyThrProValLeuLysAspIleAsnPheLysIle    435440445    GluArgGlyGlnLeuLeuAlaValAlaGlySerThrGlyAlaGlyLys    450455460    ThrSerLeuLeuMetMetIleMetGlyGluLeuGluProSerGluGly    465470475480    LysIleLysHisSerGlyArgIleSerPheCysSerGlnPheSerTrp    485490495    IleMetProGlyThrIleLysGluAsnIleIlePheGlyValSerTyr    500505510    AspGluTyrArgTyrArgSerValIleLysAlaCysGlnLeuGluGlu    515520525    AspIleSerLysPheAlaGluLysAspAsnIleValLeuGlyGluGly    530535540    GlyIleThrLeuSerGlyGlyGlnArgAlaArgIleSerLeuAlaArg    545550555560    AlaValTyrLysAspAlaAspLeuTyrLeuLeuAspSerProPheGly    565570575    TyrLeuAspValLeuThrGluLysGluIlePheGluSerCysValCys    580585590    LysLeuMetAlaAsnLysThrArgIleLeuValThrSerLysMetGlu    595600605    HisLeuLysLysAlaAspLysIleLeuIleLeuHisGluGlySerSer    610615620    TyrPheTyrGlyThrPheSerGluLeuGlnAsnLeuGlnProAspPhe    625630635640    SerSerLysLeuMetGlyCysAspSerPheAspGlnPheSerAlaGlu    645650655    ArgArgAsnSerIleLeuThrGluThrLeuHisArgPheSerLeuGlu    660665670    GlyAspAlaProValSerTrpThrGluThrLysLysGlnSerPheLys    675680685    GlnThrGlyGluPheGlyGluLysArgLysAsnSerIleLeuAsnPro    690695700    IleAsnSerIleArgLysPheSerIleValGlnLysThrProLeuGln    705710715720    MetAsnGlyIleGluGluAspSerAspGluProLeuGluArgArgLeu    725730735    SerLeuValProAspSerGluGlnGlyGluAlaIleLeuProArgIle    740745750    SerValIleSerThrGlyProThrLeuGlnAlaArgArgArgGlnSer    755760765    ValLeuAsnLeuMetThrHisSerValAsnGlnGlyGlnAsnIleHis    770775780    ArgLysThrThrAlaSerThrArgLysValSerLeuAlaProGlnAla    785790795800    AsnLeuThrGluLeuAspIleTyrSerArgArgLeuSerGlnGluThr    805810815    GlyLeuGluIleSerGluGluIleAsnGluGluAspLeuLysGluCys    820825830    LeuPheAspAspMetGluSerIleProAlaValThrThrTrpAsnThr    835840845    TyrLeuArgTyrIleThrValHisLysSerLeuIlePheValLeuIle    850855860    TrpCysLeuValIlePheLeuAlaGluValAlaAlaSerLeuValVal    865870875880    LeuTrpLeuLeuGlyAsnThrProLeuGlnAspLysGlyAsnSerThr    885890895    HisSerArgAsnAsnSerTyrAlaValIleIleThrSerThrSerSer    900905910    TyrTyrValPheTyrIleTyrValGlyValAlaAspThrLeuLeuAla    915920925    MetGlyPhePheArgGlyLeuProLeuValHisThrLeuIleThrVal    930935940    SerLysIleLeuHisHisLysMetLeuHisSerValLeuGlnAlaPro    945950955960    MetSerThrLeuAsnThrLeuLysAlaGlyGlyIleLeuAsnArgPhe    965970975    SerLysAspIleAlaIleLeuAspAspLeuLeuProLeuThrIlePhe    980985990    AspPheIleGlnLeuLeuLeuIleValIleGlyAlaIleAlaValVal    99510001005    AlaValLeuGlnProTyrIlePheValAlaThrValProValIleVal    101010151020    AlaPheIleMetLeuArgAlaTyrPheLeuGlnThrSerGlnGlnLeu    1025103010351040    LysGlnLeuGluSerGluGlyArgSerProIlePheThrHisLeuVal    104510501055    ThrSerLeuLysGlyLeuTrpThrLeuArgAlaPheGlyArgGlnPro    106010651070    TyrPheGluThrLeuPheHisLysAlaLeuAsnLeuHisThrAlaAsn    107510801085    TrpPheLeuTyrLeuSerThrLeuArgTrpPheGlnMetArgIleGlu    109010951100    MetIlePheValIlePhePheIleAlaValThrPheIleSerIleLeu    1105111011151120    ThrThrGlyGluGlyGluGlyArgValGlyIleIleLeuThrLeuAla    112511301135    MetAsnIleMetSerThrLeuGlnTrpAlaValAsnSerSerIleAsp    114011451150    ValAspSerLeuMetArgSerValSerArgValPheLysPheIleAsp    115511601165    MetProThrGluGlyLysProThrLysSerThrLysProTyrLysAsn    117011751180    GlyGlnLeuSerLysValMetIleIleGluAsnSerHisValLysLys    1185119011951200    AspAspIleTrpProSerGlyGlyGlnMetThrValLysAspLeuThr    120512101215    AlaLysTyrThrGluGlyGlyAsnAlaIleLeuGluAsnIleSerPhe    122012251230    SerIleSerProGlyGlnArgValGlyLeuLeuGlyArgThrGlySer    123512401245    GlyLysSerThrLeuLeuSerAlaPheLeuArgLeuLeuAsnThrGlu    125012551260    GlyGluIleGlnIleAspGlyValSerTrpAspSerIleThrLeuGln    1265127012751280    GlnTrpArgLysAlaPheGlyValIleProGlnLysValPheIlePhe    128512901295    SerGlyThrPheArgLysAsnLeuAspProTyrGluGlnTrpSerAsp    130013051310    GlnGluIleTrpLysValAlaAspGluValGlyLeuArgSerValIle    131513201325    GluGlnPheProGlyLysLeuAspPheValLeuValAspGlyGlyCys    133013351340    ValLeuSerHisGlyHisLysGlnLeuMetCysLeuAlaArgSerVal    1345135013551360    LeuSerLysAlaLysIleLeuLeuLeuAspGluProSerAlaHisLeu    136513701375    AspProValThrTyrGlnIleIleArgArgThrLeuLysGlnAlaPhe    138013851390    AlaAspCysThrValIleLeuCysGluHisArgIleGluAlaMetLeu    139514001405    GluCysGlnGlnPheLeuValIleGluGluAsnLysValArgGlnTyr    141014151420    AspSerIleGlnLysLeuLeuAsnGluArgSerLeuPheArgGlnAla    1425143014351440    IleSerProSerAspArgValLysLeuPheProHisArgAsnSerSer    144514501455    LysCysLysSerLysProGlnIleAlaAlaLeuLysGluGluThrGlu    146014651470    GluGluValGlnAspThrArgLeu    14751480    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5635 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGT60    TTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGT120    GATGTTGCAAGTGTGGCGGAACACATGTAAGCGCCGGATGTGGTAAAAGTGACGTTTTTG180    GTGTGCGCCGGTGTATACGGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAG240    TAAATTTGGGCGTAACCAAGTAATGTTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGA300    AGTGAAATCTGAATAATTCTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGG360    GACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTC420    CGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCTGACGCGCAGTGTATTTATACCCGG480    TGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCTCCTCCGAGCCGC540    TCCGAGCTAGTAACGGCCGCCAGTGTGCTGCAGATATCAAAGTCGACGGTACCCGAGAGA600    CCATGCAGAGGTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGA660    CCAGACCAATTTTGAGGAAAGGATACAGACAGCGCCTGGAATTGTCAGACATATACCAAA720    TCCCTTCTGTTGATTCTGCTGACAATCTATCTGAAAAATTGGAAAGAGAATGGGATAGAG780    AGCTGGCTTCAAAGAAAAATCCTAAACTCATTAATGCCCTTCGGCGATGTTTTTTCTGGA840    GATTTATGTTCTATGGAATCTTTTTATATTTAGGGGAAGTCACCAAAGCAGTACAGCCTC900    TCTTACTGGGAAGAATCATAGCTTCCTATGACCCGGATAACAAGGAGGAACGCTCTATCG960    CGATTTATCTAGGCATAGGCTTATGCCTTCTCTTTATTGTGAGGACACTGCTCCTACACC1020    CAGCCATTTTTGGCCTTCATCACATTGGAATGCAGATGAGAATAGCTATGTTTAGTTTGA1080    TTTATAAGAAGACTTTAAAGCTGTCAAGCCGTGTTCTAGATAAAATAAGTATTGGACAAC1140    TTGTTAGTCTCCTTTCCAACAACCTGAACAAATTTGATGAAGGACTTGCATTGGCACATT1200    TCGTGTGGATCGCTCCTTTGCAAGTGGCACTCCTCATGGGGCTAATCTGGGAGTTGTTAC1260    AGGCGTCTGCCTTCTGTGGACTTGGTTTCCTGATAGTCCTTGCCCTTTTTCAGGCTGGGC1320    TAGGGAGAATGATGATGAAGTACAGAGATCAGAGAGCTGGGAAGATCAGTGAAAGACTTG1380    TGATTACCTCAGAAATGATTGAAAACATCCAATCTGTTAAGGCATACTGCTGGGAAGAAG1440    CAATGGAAAAAATGATTGAAAACTTAAGACAAACAGAACTGAAACTGACTCGGAAGGCAG1500    CCTATGTGAGATACTTCAATAGCTCAGCCTTCTTCTTCTCAGGGTTCTTTGTGGTGTTTT1560    TATCTGTGCTTCCCTATGCACTAATCAAAGGAATCATCCTCCGGAAAATATTCACCACCA1620    TCTCATTCTGCATTGTTCTGCGCATGGCGGTCACTCGGCAATTTCCCTGGGCTGTACAAA1680    CATGGTATGACTCTCTTGGAGCAATAAACAAAATACAGGATTTCTTACAAAAGCAAGAAT1740    ATAAGACATTGGAATATAACTTAACGACTACAGAAGTAGTGATGGAGAATGTAACAGCCT1800    TCTGGGAGGAGGGATTTGGGGAATTATTTGAGAAAGCAAAACAAAACAATAACAATAGAA1860    AAACTTCTAATGGTGATGACAGCCTCTTCTTCAGTAATTTCTCACTTCTTGGTACTCCTG1920    TCCTGAAAGATATTAATTTCAAGATAGAAAGAGGACAGTTGTTGGCGGTTGCTGGATCCA1980    CTGGAGCAGGCAAGACTTCACTTCTAATGATGATTATGGGAGAACTGGAGCCTTCAGAGG2040    GTAAAATTAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCCTGGATTATGCCTG2100    GCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACAGAAGCG2160    TCATCAAAGCATGCCAACTAGAAGAGGACATCTCCAAGTTTGCAGAGAAAGACAATATAG2220    TTCTTGGAGAAGGTGGAATCACACTGAGTGGAGGTCAACGAGCAAGAATTTCTTTAGCAA2280    GAGCAGTATACAAAGATGCTGATTTGTATTTATTAGACTCTCCTTTTGGATACCTAGATG2340    TTTTAACAGAAAAAGAAATATTTGAAAGCTGTGTCTGTAAACTGATGGCTAACAAAACTA2400    GGATTTTGGTCACTTCTAAAATGGAACATTTAAAGAAAGCTGACAAAATATTAATTTTGC2460    ATGAAGGTAGCAGCTATTTTTATGGGACATTTTCAGAACTCCAAAATCTACAGCCAGACT2520    TTAGCTCAAAACTCATGGGATGTGATTCTTTCGACCAATTTAGTGCAGAAAGAAGAAATT2580    CAATCCTAACTGAGACCTTACACCGTTTCTCATTAGAAGGAGATGCTCCTGTCTCCTGGA2640    CAGAAACAAAAAAACAATCTTTTAAACAGACTGGAGAGTTTGGGGAAAAAAGGAAGAATT2700    CTATTCTCAATCCAATCAACTCTATACGAAAATTTTCCATTGTGCAAAAGACTCCCTTAC2760    AAATGAATGGCATCGAAGAGGATTCTGATGAGCCTTTAGAGAGAAGGCTGTCCTTAGTAC2820    CAGATTCTGAGCAGGGAGAGGCGATACTGCCTCGCATCAGCGTGATCAGCACTGGCCCCA2880    CGCTTCAGGCACGAAGGAGGCAGTCTGTCCTGAACCTGATGACACACTCAGTTAACCAAG2940    GTCAGAACATTCACCGAAAGACAACAGCATCCACACGAAAAGTGTCACTGGCCCCTCAGG3000    CAAACTTGACTGAACTGGATATATATTCAAGAAGGTTATCTCAAGAAACTGGCTTGGAAA3060    TAAGTGAAGAAATTAACGAAGAAGACTTAAAGGAGTGCCTTTTTGATGATATGGAGAGCA3120    TACCAGCAGTGACTACATGGAACACATACCTTCGATATATTACTGTCCACAAGAGCTTAA3180    TTTTTGTGCTAATTTGGTGCTTAGTAATTTTTCTGGCAGAGGTGGCTGCTTCTTTGGTTG3240    TGCTGTGGCTCCTTGGAAACACTCCTCTTCAAGACAAAGGGAATAGTACTCATAGTAGAA3300    ATAACAGCTATGCAGTGATTATCACCAGCACCAGTTCGTATTATGTGTTTTACATTTACG3360    TGGGAGTAGCCGACACTTTGCTTGCTATGGGATTCTTCAGAGGTCTACCACTGGTGCATA3420    CTCTAATCACAGTGTCGAAAATTTTACACCACAAAATGTTACATTCTGTTCTTCAAGCAC3480    CTATGTCAACCCTCAACACGTTGAAAGCAGGTGGGATTCTTAATAGATTCTCCAAAGATA3540    TAGCAATTTTGGATGACCTTCTGCCTCTTACCATATTTGACTTCATCCAGTTGTTATTAA3600    TTGTGATTGGAGCTATAGCAGTTGTCGCAGTTTTACAACCCTACATCTTTGTTGCAACAG3660    TGCCAGTGATAGTGGCTTTTATTATGTTGAGAGCATATTTCCTCCAAACCTCACAGCAAC3720    TCAAACAACTGGAATCTGAAGGCAGGAGTCCAATTTTCACTCATCTTGTTACAAGCTTAA3780    AAGGACTATGGACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACTCTGTTCCACA3840    AAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCC3900    AAATGAGAATAGAAATGATTTTTGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTT3960    TAACAACAGGAGAAGGAGAAGGAAGAGTTGGTATTATCCTGACTTTAGCCATGAATATCA4020    TGAGTACATTGCAGTGGGCTGTAAACTCCAGCATAGATGTGGATAGCTTGATGCGATCTG4080    TGAGCCGAGTCTTTAAGTTCATTGACATGCCAACAGAAGGTAAACCTACCAAGTCAACCA4140    AACCATACAAGAATGGCCAACTCTCGAAAGTTATGATTATTGAGAATTCACACGTGAAGA4200    AAGATGACATCTGGCCCTCAGGGGGCCAAATGACTGTCAAAGATCTCACAGCAAAATACA4260    CAGAAGGTGGAAATGCCATATTAGAGAACATTTCCTTCTCAATAAGTCCTGGCCAGAGGG4320    TGGGCCTCTTGGGAAGAACTGGATCAGGGAAGAGTACTTTGTTATCAGCTTTTTTGAGAC4380    TACTGAACACTGAAGGAGAAATCCAGATCGATGGTGTGTCTTGGGATTCAATAACTTTGC4440    AACAGTGGAGGAAAGCCTTTGGAGTGATACCACAGAAAGTATTTATTTTTTCTGGAACAT4500    TTAGAAAAAACTTGGATCCCTATGAACAGTGGAGTGATCAAGAAATATGGAAAGTTGCAG4560    ATGAGGTTGGGCTCAGATCTGTGATAGAACAGTTTCCTGGGAAGCTTGACTTTGTCCTTG4620    TGGATGGGGGCTGTGTCCTAAGCCATGGCCACAAGCAGTTGATGTGCTTGGCTAGATCTG4680    TTCTCAGTAAGGCGAAGATCTTGCTGCTTGATGAACCCAGTGCTCATTTGGATCCAGTAA4740    CATACCAAATAATTAGAAGAACTCTAAAACAAGCATTTGCTGATTGCACAGTAATTCTCT4800    GTGAACACAGGATAGAAGCAATGCTGGAATGCCAACAATTTTTGGTCATAGAAGAGAACA4860    AAGTGCGGCAGTACGATTCCATCCAGAAACTGCTGAACGAGAGGAGCCTCTTCCGGCAAG4920    CCATCAGCCCCTCCGACAGGGTGAAGCTCTTTCCCCACCGGAACTCAAGCAAGTGCAAGT4980    CTAAGCCCCAGATTGCTGCTCTGAAAGAGGAGACAGAAGAAGAGGTGCAAGATACAAGGC5040    TTTAGAGAGCAGCATAAATGTTGACATGGGACATTTGCTCATGGAATTGGAGGTAGCGGA5100    TTGAGGTACTGAAATGTGTGGGCGTGGCTTAAGGGTGGGAAAGAATATATAAGGTGGGGG5160    TCTCATGTAGTTTTGTATCTGTTTTGCAGCAGCCGCCGCCATGAGCGCCAACTCGTTTGA5220    TGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCA5280    GAATGTGATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTT5340    GACCTACGAGACCGTGTCTGGAACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGC5400    CGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAG5460    CAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATT5520    GGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCA5580    GGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAA5635    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ACTCTTGAGTGCCAGCGAGTAGAGTTTTCTCCTCCG36    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GCAAAGGAGCGATCCACACGAAATGTGCC29    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CTCCTCCGAGCCGCTCCGAGCTAG24    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    CCAAAAATGGCTGGGTGTAGGAGCAGTGTCC31    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CGGATCCTTTATTATAGGGGAAGTCCACGCCTAC34    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CGGGATCCATCGATGAAATATGACTACGTCCG32    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 170 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GTACGGTTGATCTTCTCCATTCCCCGAGTGGTCAAGTTTTAGACTTCACCTCTGTCCTGG60    ACTCCACTGTTACTGTAGATGAGACTGTAAGAGAGGAGTCCTGTAGAGGTTCAAACGTCT120    CTTTCTGTTATATCAAGAACCTCTTCCACCTTAGTGTGACTCACCTCCAG170    __________________________________________________________________________

We claim:
 1. An adenoviral vector comprising an adenovirus genome fromwhich the E1, E2, E3 and E4 regions and late genes of the adenovirusgenome have been deleted and additionally comprising a nucleic acid ofinterest operably linked to expression control sequences.
 2. The vectorof claim 1 wherein the vector comprises the 5' and 3' inverted terminalrepeat sequences, 5' packaging sequences and the E1A enhancer sequencesof the adenovirus genome.
 3. The vector of claim 2 wherein the vectorfurther comprises a nuclear localization signal and a polyadenylationsignal.
 4. The vector of claim 3 wherein the expression controlsequences comprise a PGK promoter.
 5. The vector of claim 3 wherein theexpression control sequences comprise the adenovirus major late promoterand the adenovirus tripartite leader, the nuclear localization signalcomprises the SV40 T-antigen nuclear localization signal and thepolyadenylation signal comprises the SV40 polyadenylation signal.
 6. Thevector of claim 5 wherein the vector further comprises an internalribosomal entry site.
 7. The vector of claim 6 wherein the internalinitiation signal is the EMC internal ribosomal entry signal.
 8. Anadenoviral vector comprising the 5' and 3' inverted terminal repeat(ITR) sequences, the 5' packaging sequences and the E1A enhancersequences of the adenovirus genome, wherein the vector can accommodate aforeign nucleic acid of up to 36 kb in length.
 9. The vector of claim 8further comprising a foreign nucleic acid of interest operably linked toexpression control sequences.
 10. The vector of claim 8 wherein the 5'inverted terminal repeat sequences, the 5' packaging sequences and theE1A enhancer sequence comprise nucleotides 0-358 of the adenoviralgenome.
 11. The vector of claim 8 wherein the 5' inverted terminalrepeat sequences, the 5' packaging sequences and the E1A enhancersequence comprise nucleotides 0-380 of the adenoviral genome.
 12. A PAVI vector.
 13. A PAV II vector.
 14. A method for producing an adenoviralvector comprising an adenovirus genome from which the E1, E2, E3 and E4regions and late genes of the adenovirus genome have been deleted andadditionally comprising a foreign nucleic acid of interest operablylinked to expression control sequences, the method comprisingintroducing the adenoviral vector nucleic acid and a helper virusnucleic acid into an adenovirus producer cell line and purifying theadenoviral vector and helper virus from said cell line.
 15. A method forproducing an adenoviral vector comprising the 5' and 3' invertedterminal repeat (ITR) sequences, the 5' packaging sequences and the E1Aenhancer sequences of the adenoviral genome, wherein the vector canaccommodate a foreign nucleic acid of up to 36 kb in length, the vectoradditionally comprising a foreign nucleic acid of interest operablylinked to expression control sequences, the method comprisingintroducing the adenoviral vector nucleic acid and a helper virusnucleic acid into an adenovirus producer cell line and purifying theadenoviral vector and helper virus from said cell line.
 16. The methodof claim 14 or 15, further comprising purifying the adenoviral vector bycesium chloride gradient centrifugation.
 17. The method of claim 14 or15, wherein the helper virus supplies adenovirus replication and/oradenoviral structural proteins to the adenoviral vector, therebyallowing said adenoviral vector to be replicated and packaged.
 18. Themethod of claim 17, wherein the helper virus is a packaging and/orreplication deficient adenovirus.
 19. The method of claim 17, whereinthe helper virus is a packaging defective adenovirus.
 20. The method ofclaim 19 wherein the helper virus has mutations in the adenoviruspackaging sequences.
 21. The method of claim 17, wherein the helpervirus is a replication deficient adenovirus.